Title of Invention	"A NANOCOCHLEATE -NANOSPHERE COMPLEX"
Abstract	This invention relates to a nano-complex comprising (i) nanospheres with a first drug entrapped within said nanospheres and (ii) lipid based nanocochleates with a second drug encapsulated therein, characterized in that, said nanospheres are chemically or physically linked either within or on the surface of the nanocochleate such that a combination of drugs are co-encapsulated in a single nanosphere-nanocochleate complex. This invention also relates to a process for preparation of such nano-complex.

Title of Invention

"A NANOCOCHLEATE -NANOSPHERE COMPLEX"

Abstract

This invention relates to a nano-complex comprising (i) nanospheres with a first drug entrapped within said nanospheres and (ii) lipid based nanocochleates with a second drug encapsulated therein, characterized in that, said nanospheres are chemically or physically linked either within or on the surface of the nanocochleate such that a combination of drugs are co-encapsulated in a single nanosphere-nanocochleate complex. This invention also relates to a process for preparation of such nano-complex.

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 of 1970) AND THE PATENTS RULES, 2003 COMPLETE SPECIFICATION (See Section 10; rule 13) TITLE " A NANOCOCHLEATE-NANOSPHERE COMPLEX " APPLICANT Indian Institute of Technology, Bombay, Dept. of Biosciences and Bioengineering, 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 THE INVENTION: The present invention is directed towards development of nano-complexes comprising nanococleates linked with nanospheres for delivery of two or more drugs. The nanospheres are either chemically or physically bound to the nanocochleates by a simplified procedure which avoids the use of synthetic polymers. Thus, another object of the invention relates to a process for preparing nanocochleate-nanosphere complexes using nanospheres of biodegradable biopolymers and proteins rather than cationic molecules for delivery of hydrophobic and hydrophilic drugs. BACKGROUND OF THE INVENTION: Many diseases require therapy with two or more drugs, combinations of the drugs in such a manner as to allow sustained release and reduce the frequency of doses is desirable. One such condition where this would be particularly useful is in cancers. Cancer is the second most common cause of death among adults in most countries. Great progress has been made in the treatment of selected malignancies and approximately 50% of all malignancies can be treated using current treatment strategies. Still, majority of these treatments are carried out by surgery. However, surgical excision is not effective when the cancer cells have infiltrated the nearby vital organs or have spread to other parts of the body. Radiotherapy and chemotherapy used alone or in combination have also shown to greatly improve the management of patients with a variety of solid and haematologic malignancies. Chemotherapeutic drugs may destroy healthy tissue along with the cancer cells and also give rise to adverse side effects such as fatigue, vomiting, alopecia, gastro-intestinal disturbances, anemia etc. Further, by oral delivery of the chemotherapeutic agents about 8-10 of the active drug is either denatured by the stomach environment or eliminated via liver metabolism. Thus, despite improvements in the treatment of most metastatic solid tumors, these remain largely incurable. Reasons for this are insufficient tumor selectivity of anti-cancer agents and poor penetration within the tumor mass. To overcome the failure of conventional therapy, combination of two different cytotoxic chemotherapeutic agents is given. This combination therapy improves cancer remission rate and duration of survival. At present, combination therapy is achieved by two or more drugs being administered separately which does not allow one to achieve controlled and sustained release from the formulations. In addition, there is a need to co-encapsulate hydrophobic and hydrophilic drugs in the same carrier through non-invasive routes like oral or aerosol. In this domain, particularly for dual delivery of drugs, nanotechnology based therapeutics have given rise to enormous potential in addressing the failures of traditional therapeutics that could not be effectively formulated owing to poor water solubility or lack of target specificity. Nanoparticles are preferred for their properties such as biodegradability, biocompatibility, conjugation, encapsulation and their ability to be functionalized. Further, these nanoparticles are superior over conventional drug delivery methods as these can be targeted to a specific area and owing to their extremely small size they can penetrate through smaller capillaries and are easily taken up by cancer cells. US 7,304,045 B2 describes a dual particle tumor targeting system consisting of a galactosamine conjugated nanoparticle and a second EPR mediated nanoparticle mixed together in a solution. Also, the two nanoparticles are simply administered at different time points as there is no single composite entity formed by the interaction of the nanoparticles There is no covalent interaction between the two nanostructures, thereby making the targeting of the second nanoparticle passive. On the other hand, the present invention does not require administration of the two particles at different time points as the constituent nanospheres and nanocochleates are engineered to release the drugs at different time points. Patent No. US 7,244,781 B2 describes the method of preparation of composite compositions including polymeric nanoparticles and clay nanoparticles. The nanoparticles, being non biodegradable and non-biocompatible, would be cleared by the reticulo-endothelial system and elicit immune response by the body. US 2010/0203163 describes an injectable polymer-lipid blend provided as a localized drug delivery system for a pharmaceutically active agent. The effectiveness of the system is dependent upon the rheological properties and the viscoelasticity of the blend. Also, it is distinct from the dual-delivery system developed by virtue of being a matrix based system. US 7511023 and US 2004/0138152 describes the method of preparation of anticancer drug-chitosan complex forming self aggregates in aqueous media composed of a hydrophobic anticancer agent and a hydrophilic chitosan. The present invention deals with alginate-chitosan nanoparticles complexed with lipid nanocochleates for dual drug delivery. US 7,740,883 and US 2005/0226938 describe methods for preparing cross linked core and core-shell nanoparticle polymers from chitosan. The dual delivery system developed by us comprises of chitosan nanoparticles specifically modified with alginic acid to increase the encapsulation of hydrophilic anthracycline drugs. US 2009/0036417 and US 2004/0092727 describe cochleate and nanocochleate systems wherein the agents bridging agents are organic multi-valent cations. In the dual-delivery system developed by us, nanoparticles are used as bridging agents for formation of cochleates. US 20030185894 describes a process for producing nanoparticles of hydrophobic drug paclitaxel and albumin. In the dual delivery nanocomplex system developed by us, albumin nanoparticles are used to encapsulate the hydrophilic drug doxorubicin. US20060246524 and US20090181398 describe conjugate compositions that include a specific-binding moiety covalently coupled to a nanoparticle through a heterobifunctional polyalkyleneglycol linker. In the dual nano-complex system developed by us, two distinct species of nanoparticles are physically or chemically bound together without the use of any linker molecule. US 2009/0053297 describes negatively charged and PEGylated lipid structures (liposomes, cochleates, micelles) for reducing the immunogenicity and increasing the half life of therapeutic proteins such as Factor VIII. The system uses a single lipid structure to encapsulate therapeutic proteins. It does not deal with nanosphere-nanocochleate complexes for combination drug delivery. US Patents 4078052 and 5840707 deal with the development of cochleates from large unilamellar vesicles and for the delivery of proteins or peptides as vaccines. These patents do not deal with control of sizes in the form of nanocochleates and do not deal with nanocomplexes for dual drug delivery. US Patent 6153217 deals with a process for the preparation of nanocochleates for oral or mucosal drug delivery. It comprises of preparing small unilamellar liposomes of negatively charged lipid mainly phosphatidylserine and mixing the liposome suspension with synthetic polymer A consisting of dextran or polyethyleneglycol 2-20%w/w. The liposome/polymer suspension is then added into a solution of polymer B (wherein polymers A and B are immiscible) like polyvinylpyrolidone or polyvinylalcohol or Ficoll or polyvinylmethylether or polyethylene glycol 2-20%w/w. Finally a solution of a positively charged molecule which is a cationic salt is added to the two phase polymer system and excess polymer is removed by centrifugation. Despite the aforesaid, there is still a need for a unique nano-complex system comprising two different nanoparticle systems with better physiochemical properties, anti-tumor activity and targeting ability. Furthermore, the present invention deals with nanosphere-nanocochleate complexes in which the nanocochleates are held together by biopolymeric or protein nanospheres instead of adding cations or drugs (as used in prior art) to facilitate the acquisition of their characteristic multilayered spiral structure. Thus, the present invention deals with the development of nanocochleate-nanosphere complexes with a new process where biopolymeric and protein nanospheres are formed and directly added during the hydration of the unilamellar liposomes to form nanocochleate- nanosphere complexes for dual drug delivery (both hydrophobic and hydrophilic in nature). The invention employs alginate-chitosan and albumin nanospheres for causing precipitation of the nano-cochleate-nanosphere complexes for dual drug delivery. SUMMARY OF THE INVENTION An object of this invention is to develop biodegradable lipopolymeric and lipoprotein nanocochleate-nanosphere complexes for delivery of both hydrophobic and hydrophilic drugs. Another object is to provide sustained release, gastric resistance and synergistic action of the dual drugs encapsulated within the nanocomplexes. Another object of the invention is to provide active targeting and endocytosis of the nanocochleate-nanosphere complex. A further object of this invention is in process for preparing nanocohleate-nanosphere complexes using nanospheres of biodegradable biopolymers and proteins rather than cationic molecules, for delivery hydrophobic and hydrophilic drugs. ADVANTAGES The present invention has the following advantages over the prior art: Biodegradable biopolymeric and protein nanospheres stably linked with lipid nanocochleates - simple process for preparation which avoids cationic salts for crosslinking - size of 300-800 nm - shows high cellular internalization by endocytosis in cancer cells - stable in harsh conditions of gastric acids and enzymes delivers hydrophobic and hydrophilic drugs in a sustained manner Other features and advantages of the present invention will become apparent as the following detailed description proceeds or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings: Figure 1: A & B- Transmission electron microscopic image of chitosan alginate nanospheres; C & D- Transmission electron microscopic image of nanosphere on nanocochleate complex using chitosan alginate nanospheres Figure 2: A & B- Transmission electron microscopic image of nanosphere in nanocochleate complex using chitosan alginate nanospheres. Figure 3: A & B- Transmission electron microscopic image of albumin nanospheres; C & D- Transmission electron microscopic image of nanosphere on nanocochleate complex using albumin nanospheres Figure 4: Confocal Microscopy images of Calcein encapsulated Alginate chitosan nanospheres encochleated inside nanocochleates containing Rhodamine 6G dye (5µm) (without any drugs) Figure 5: Cellular internalization of the calcein loaded alginate chitosan nanospheres (green) B with rhodamine 6G nanocochleates (red) A in HCT-15 cancer cell line by confocal laser scanning microscopy (both the images were captured at the same location with two different laser source) without any drugs Figure 6: Cellular internalization of the Rhodamine-6G loaded albumin nanospheres (red) B with curcumin loaded nanocochleates (green) A in MDA-MB 231 cancer cell line by confocal laser scanning microscopy (both the images were captured at the same location with two different laser source) (without any drugs) Figure 7: Cellular internalization of the Rhodamine-6G loaded alginate chitosan nanospheres (red) B with curcumin loaded nanocochleates (green) A in MDA-MB 231 cancer cell line by confocal laser scanning microscopy (both the images were captured at the same location with two different laser source) (without any drugs) Figure 8: A- Paclitaxel release from nanosphere in nanocochleates complex using alginate chitosan nanospheres versus marketed standard paclitaxel formulation; B Doxorubicin release from same nanosphere- nanocochleates complex versus standard doxorubicin Figure 9: A- Paclitaxel release from nanosphere on nanocochleate complexes using alginate chitosan nanospheres versus marketed standard paclitaxel formulation; B Doxorubicin release from nanosphere-nanocochleate complex versus standard doxorubicin Figure 10: A- Paclitaxel release from nanosphere on nanocochleate complex using albumin nanospheres versus marketed standard paclitaxel formulation; B-Doxorubicin release from same nanosphere nanocochleates complex formulation versus standard doxorubicin Figure 11: Drug release in simulated gastric fluid(0.1 M Hydrochloric acid) at two different intervals DETAILED DESCRIPTION OF THE INVENTION In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section. Specific and preferred values listed below for individual components, substituents, and ranges are for illustration only; they do not exclude other defined values or other values within defined ranges for the components and substituents. As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified systems or process parameters that may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the scope of the invention in any manner. In our present invention we have encapsulated two different drugs for combination chemotherapy in two different nanoparticle systems which are combined together to forma unique nano-complex system with better physiochemical properties, anti-tumour activity and targeting ability than either of the constituents. In these regard spherical nanoparticles were prepared from biopolymers and their derivatives (such as chitosan, alginate, albumin) encapsulating drug 1(D1). These nanoparticles were used as complexing agent for the preparation of nanocochleates made up of lipids such as Phosphatidylserine (DOPS), Phosphatidylcholine (DPPC) and Cholesterol. The invention deals with nanosphere-nanocochleate complexes in which the nanocochleates are held together by biopolymeric or protein nanospheres instead of adding cations or drugs (as used in prior art) to facilitate the acquisition of their characteristic multilayered spiral structure. The anticancer drug 1 (Dl) entrapped (or adsorbed on surface) within spherical nanoparticles helps in the formation of nanocochleate like structure while the drug (D2) is encapsulated within the bilayer of the nanocochleates. Two distinct species of nano-complexes were obtained by this method depending upon the size of the spherical nanoparticles: (i) Nanosphere in nanocochleate: Spherical nanoparticles entrapped inside spiral nanocochleates for (ii) Nanosphere on Nanocohleate: Spherical nanoparticles covalently attached on the surface of the spiral nanocochleates. In context of the present invention, nanocochleates are cylindrical (cigar-like) microstructures that consist of a series of lipid bilayers which are formed as a result of the condensation of small unilamellar negatively charged liposomes. They have a unique multilayered structure consisting of a solid, lipid bilayer sheet rolled up in a spiral or in stacked sheets, with little or no internal aqueous space. Because the entire nanocochleate structure is a series of solid layers, components encapsulate within the interior of the nanocohleate structure and remain intact, even though the outer layers of the nanocochleate may be exposed to harsh environmental conditions. The drug is preferably encapsulated in the lipid bilayer of the nanocochleates. The different lipids that make up the nanocochleates include but are not limited to phosphotidyl serine [PS], dioleoylphosphatidyl serine [DOPS], phosphatidic acid [PA], phosphatidylinositol [PI], phosphatidyl glycerol [PG], phosphatidylcholine [PC], phosphatidylethanolamine [PE], diphosphoticlylglycerol [DPG], dioleoyl phosphatidic acid [DOPA], distearoyl phosphatidyl serine [DSPS], dimyristoyl phosphatidyl serine [DMPS], dipalmitoyl phosphatidylglycerol [DPPG] or cholesterol. Nanocochleates of the present invention, without intending to be limited therein are preferably made of the lipid dioleyl phosphatidylserine [DOPS]. According to a preferred embodiment of the present invention, the lipid Dioleyl phosphatidylserine (DOPS) was employed as major component for formation of the structure. DOPS is an anionic phospholipid which tends to provide negative charge so as to facilitate the assembly of the spiral multilayered structure. In addition, DOPS is a completely biocompatible and biodegradable lipid. It is, in fact, of nutritious value and has benefits in memory and learning enhancement. In addition to it cholesterol and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine) were added to improve the stability of the structure during storage as well as when administered for activity. The particle size of the nanocochleates are preferably in the range of 300 - 500 nm. In accordance to the instant invention, nanospheres are solid-state nanoparticles ranging from 10 to 200 nm in size. These are mainly spherical particles which can be either amorphous or crystalline in nature and have the ability to protect the drug from enzymatic and chemical degradation. The drug is dissolved or encapsulated or attached to the nanoparticles and, depending on the methods employed for its preparation; one can obtain nanospheres, nanocapsules or aquasomes. Nanospheres are generally composed of natural polymers such as chitosan, gum, albumin, gelatin or collagen and the drug is uniformly dispersed in it. The tiny capsule of drug store house is called vesicle and the solid skeleton structure is called nanosphere. The nanospheres of the present invention are biodegradable in nature. The particle size of the nanospheres are preferably in the range of 40 - 100 nm. Nano-complexes meant for dual delivery of pharmaceutically active entities in accordance to the present invention comprises nanospheres linked with nanocochleates wherein the nanosphere is either entrapped inside the nanocochleate or covalently attached on the surface of the nanocochleate. Thus, nano-complexes meant for dual delivery of pharmaceutically active entities consisting of lipid nanocochleates (particle size 300-500 nm) either incorporating or covalently attaching comparatively smaller spherical nanoparticles on their surfaces (particle size 40-100 nm). The active entities are encapsulated into the nanocochleates and the nanospheres, thereby ensuring simultaneous delivery of both the pharmaceuticals for combination chemotherapy. Doxorubicin and paclitaxel were the drugs used as the representative hydrophobic and hydrophilic drugs encapsulated within the nano-complexes. This formulation prepared intended for different routes of administration from oral to aerosol. Therefore, the present developed formulation comprising two different anticancer drugs tend to show good anticancer activity than when it is administered separately in multidrug resistant cells. One aspect of the invention relates to a nano-complex comprising nanospheres with a first drug entrapped within said nanospheres and lipid based nanocochleates with a second drug encapsulated therein, characterized in that, said nanospheres are chemically or physically linked either within or on the surface of the nanocochleate such that a combination of drugs are co-encapsulated in a single nanosphere-nanocochleate complex. The present invention however contemplates the use of two or more drugs for encapsulation and delivery. More specifically, the present invention is a nano-complex delivery vehicle comprising of a nanocochleate formulation associated with smaller spherical nanoparticles, which in turn, also serve as complexing agent for nanocochleate formation. These nanostructures comprise of two different anti-cancer drugs for effective treatment of the cancer by combination chemotherapy. The spherical nanoparticles (made up of biopolymers such as alginate, chitosan, albumin) contain the hydrophilic anticancer drug and the other hydrophobic anticancer drug is encapsulated in the lipid bilayer of the nanocochleates. Thus two different anticancer drugs are co-encapsulated in one single nano-complex thereby facilitating synergistic anticancer activity. The present invention although explains the use of nano-complexes for dual delivery of drugs for combination chemotherapy but the scope is not restricted to anticancer agents per se. The drug may be an anti-cancer agent, anti-viral agent , an anesthetic, anti-infectious, steroidal anti-inflammatory, anti-fungal, non-steroidal anti-inflammatory, tranquiliser, nutritional supplement, vasodilatory agent and the like. These nanostructures also help various other pharmaceutically active entities to cross cell barriers, permit solubilization and protects compounds during transport owing to their unique physiochemical properties. EXAMPLES The following examples are provided to better illustrate the claimed invention and are not to be interpreted in any way as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. EXAMPLES EXAMPLE 1: PREPARATION OF NANOSPHERE IN NANOCOCHLEATE COMPLEXES USING ALGINATE CHITOSAN NANOSPHERES PREPARATION OF ALGINATE CHITOSAN NANOSPHERES (CALG-1) Alginate chitosan nanoparticles were prepared using controlled gelification of alginate in presence of cation and chitosan. Briefly, calcium chloride (0.5 ml, 18mM) was added to 9.5 ml of sodium alginate solution (0.06% w/v) containing drug. 2 ml of chitosan solution (0.05% w/v) was added followed by stirring for 30 min and the mixture was kept overnight at room temperature. Drug loaded nanoparticles were recovered by centrifugation at 32000 rpm for 30-45 min and washed thrice with TES buffer to obtain the final pellet. The supernatant solution was used to determine the concentration of unentrapped drug (doxorubicin (Dl)'by using fluorescence spectrometer with excitation and emission wavelength of 470nm and 580 nm. PREPARATION OF NANOCOCHLEATES ENCAPSULATING ALGINATE-CHITOSAN NANOSPHERES (NANOSPHERE-IN NANOCOHLEATE) (CAL-DP 1) Dioleyl Phosphatidylserine (DOPS) and cholesterol in the ratio of 9:1 were dissolved in chloroform-methanol (2:1). Thin film of lipids was formed using rotary evaporator at 40°C under vacuum. For drug-D2 (paclitaxel) encapsulation, drug dissolved in methanol and added along with lipids during film formation step. Then film was hydrated with nanoparticles in TES-Buffer to get lipid concentration of 10mg lipid/ml at 45°C for 20 min and sonicated for a minute using probe sonicator. Thus encochleated nanoparticles centrifuged at 25,000g for 10 min to separate un-encapsulated nanoparticles. The pellet reconstituted accordingly with TES Buffer containing 0.5mM of calcium chloride. EXAMPLE 2: PREPARATION OF NANOSPHERE ON NANOCOCHLEATE COMPLEXES USING ALGINATE_CHITOSAN NANOSPHERES 2.1. PREPARATION OF ALGINATE-CHITOSAN NANOPARTICLES AS DESCRIBED IN EXAMPLE 1 2.2. PREPARATION OF NANOCOCHLEATES WITH COVALENTLY ATTACHED ALGINATE-CHITOSAN NANOPARTICLES (CAL-DP2) For the preparation of the dual delivery nano-complex, a cochleate suspension comprising the lipids DOPS and DPPC (in the ratio 4:1) and encapsulating drug D2 (paclitaxel) was prepared by the method stated above in example 1(b). The aliquot of the alginic acid modified chitosan nanoparticle solution was added during the film hydration step. 100mg l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and 50mg N-hydroxysuccinimide (NHS) were also added to the cochleate/chitosan-alginate mixture to facilitate covalent bonding between the surface functional groups of nanocochleates and alginate-chitosan nanoparticles. The carbodiimide was solubilized using a vortex mixture. The mixture was reacted for 2h at room temperature (6) and was centrifuged. The supernatant of the centrifuged solution was discarded and the pellet was re-suspended in 10 ml of TES buffer. The resultant suspension was then sonicated and then lyophilized. EXAMPLE 3: PREPARATION OF NANOSPHERE ON NANOCOCHLEATE COMPLEXES USING ALBUMIN NANOSPHERES 3.1. PREPARATION OF ALBUMIN NANOPARTICLES (ALB) For the preparation of albumin nanoparticles, aliquots of 4.0 mg of doxorubicin and 20 mg of Bovine Serum Albumin (BSA) were dissolved in 1ml of purified water and stirred for 2 h. 3ml of ethanol was added drop wise to facilitate the self assembly of albumin nanoparticles. Further it was crosslinked using aqueous 8% glutaraldehyde solution (0.294-1.175µl/mg BSA, 24 h). The pH was adjusted to 8.2 after carrying out the desolvation step at pH 6.5. Further particles were purified by centrifugation at 25000g for 20 minutes, and then dispersed in water and lyophilized. The supernatant solution was used to determine the concentration of un-entrapped doxorubicin (Dl) by using fluorescence spectrometer with excitation and emission wavelength of 480nm and 550 nm. 3.2. PREPARATION OF NANOCOCHLEATES WITH COVALENTLY ATTACHED ALBUMIN NANOPARTICLES (CALB-DP) For the preparation of the dual delivery nano-complex, a cochleate suspension comprising the lipids DOPS and DPPC (in the ratio 4:1) and encapsulating drug D2 (paclitaxel) was prepared by the method stated above in example 1(b). The aliquot of the albumin nanoparticle solution was added during the film hydration step. 100mg EDC and 50mg N-hydroxy succinimide (NHS) were also added to the cochleate/albumin mixture to facilitate covalent bonding between the surface functional groups of nanocochleates and albumin nanoparticles. The carbodiimide was solubilized using a vortex mixture. The mixture was reacted for 2h at room temperature and was centrifuged. The supernatant of the centrifuged solution was discarded and the pellet was re-suspended in 10 ml of TES buffer. The resultant suspension was then sonicated. The resultant suspension was lyophilized and FT-IR spectroscopy was carried out. EXAMPLE 4 Nanosphere-nanocochleate systems as described in example 1 wherein the method of alginate-chitosan nanosphere formation is slightly modified: About 1% chitosan solution was prepared in 1% acetic acid. 1% TPP solution was prepared in water and an equivalent amount of alginic acid was added to it and the mixture was incubated overnight in the dark. Nanoparticles were spontaneously obtained under constant stirring at room temperature for 1 hour at 1000 rpm. The prepared nanoparticles were separated by centrifugation at 25000g for 20 minutes, and then dispersed in water and lyophilized, followed by steps for incorporation in nanocochleates as described in example 1. EXAMPLE 5: 5.1. CHARACTERISATION OF NANOSPHERE NANOCOCHLEATE COMPLEXES The typical structure of the nanoparticles and nanocochleates was confirmed by using electron microscopy (Transmission and scanning electron microscopy). Particle size measured using dynamic light scattering (Table 1) were in coherence with particles size as observed in electron microscopy studies (Figure 2, Figure 1, Figure 1). The Zeta potential of the different formulations is stated in Table 1. TABLE 1: PHYSIOCHEMICAL PROPERTIES OF DIFFERENT FORMULATIONS: Formulation Particle size(nm) Polydispersity index Zeta potential (mV) Alginate chitosan 32.7 ±4.6 0.14 ±0.29 -14.7 ±6.9 nanospheres Nanosphere in 482.3 ±223.1 0.32 ±0.18 -72.8 ±11.4 nanocochleate using chitosan alginate nanospheres Nanosphere on 836.6 ±199.1 0.45 ±0.12 -20.9 ±1.5 nanocochleate using chitosan alginate nanosphere Albumin nanospheres 280.3 ± 8.2 0.14± 0.31 -16±2.6 Nanosphere on 519.4 ±45.2 0.21 ±0.20 -31 ±3.5 nanocochleates using albumin nanospheres Further to confirm the nanoparticle in cochleate. confocal microscopy of the same was carried out with two different dyes (cresol red and calcein). Rhodamine 6G (lmg/ml) was dissolved in ethanol and added along with lipids during thin film formation. On the other hand Calcein loaded nanoparticles were prepared separately and nanocochleates were prepared out of them as explained in example 1,2,3.The confocal microscopy images confirmed the formation of needle shaped cochleate like structure (Figure 4) with two different dyes. 5.2. ENCAPSULATION AND IN VITRO RELEASE OF DRUGS FROM NANOSPHERE-NANOCOCHLEATECOMPLEXES The invented formulation showed high encapsulation of paclitaxel (Table 2). Similarly, high entrapment of hydrophilic drug such as doxorubicin was observed (Table 2). TABLE 2: ENCAPSULATION EFFICIENCY OF DRUGS Encapsulation efficiency (%) Drug Paclitaxel Doxorubicin CAL-DP-1 81.9±7.7 66.2 ±2.2 CAL-DP-2 76.8 ±4.3 73.8 ±0.3 CALB-DP 79.3 ±6.1 89.2 ± 0.4 In vitro release was performed for two different drugs using dialysis bag method in 25% methanolic phosphate buffer (pH 7.4). Methanol was incorporated in the media in order to overcome the solubility issue of the drug. 5.2.1. CAL-DP1: Approximately 30% paclitaxel was released from cochleates into release media over 48 h of dialysis where as standard PTX released 85% with in 3h shows sustained and controlled release of drug from the cochleates (Figure 8). Similarly, doxorubicin as estimated by fluorescence spectrometer (R2 = 0.989) was found to be released 76% of the total drug in 24h in comparison to the free standard doxorubicin released similar quantity in6 0 min explains the controlling release nature of the two drugs from the formulation (Figure 8). To test the cochleate formulation for oral delivery we tried to identify the gastric resistance of the same in 25% methanolic 0.1M hydrochloric acid (pH 1.2) by in vitro release using dialysis bag method for an hour. Paclitaxel and Doxorubicin released only 10% and 37% from the cochleate respectively and found to exhibit gastric resistance (Figure 11). Thus this formulation can also be administered via oral route. 5.2.2. CAL-DP2: The percentage of Paclitaxel released from cochleates was about 22% in 26 hours (Figure 9). Doxorubicin as estimated by fluorescence spectrometer (R2 = 0.989) was found to be released 60% in 26h (Figure 9). There was a marked improvement in the control of release of Doxorubicin from chitosan alginate nanoparticles covalently attached to nanocochleates as compared to the free standard doxorubicin. This formulation can also be administered via oral route owing to its controlled release and gastric resistance as shown by the reduction in release of both paclitaxel and doxorubicin at acidic pH (Figure 11). 5.2.3. CALB-DP: The percentage of Paclitaxel released from cochleates was about 19% in 72 hours (Figure 10). Doxorubicin as estimated by fluorescence spectrometer (R2 = 0.989) was found to be released 60% in 72h (Figure 10). There was a marked improvement in the control of release of Doxorubicin from albumin nanoparticles covalently attached to nanocochleates as compared to the free standard doxorubicin. This formulation can be considered for intravenous delivery. Oral administration is also possible owing to the presence of specific uptake pathways for albumin, a natural nutrient for the body EXAMPLE 6: SYNERGISTIC ACTION OF DUAL DRUGS LOADED WITHIN NANOSPHERE_NANOCOHLEATE COMPLEXES In vitro anti-cancer activity - The formulations were tested for cytotoxicity in the following cell lines: CAL-DP 1: HeLa (drug sensitive cell line), HCT-15(multi drug resistant cancer cell line) CAL-DP 2 and CALB-DP: MDA-MB 231 (multidrug resistant cell line) The IC-50 values are shown in the Table 3. As compared to PTX or DOX alone, the combination index calculated as explained by chou et al showed synergistic activity. Thus two drug combination within the cochleate showed synergistic effect in killing the multi¬drug resistant cancer cell line. TABLE 3: INHIBITORY CONCENTRATION (IC50 nM) OF ANTICANCER (PACLITAXEL) FORMULATION ON DIFFERENT CANCER CELL LINES (n=3) IC50 (nM) Cancer cell Formulation PTX DOX PTX+DOX Combination Interaction line index type Human CAL-DP1 12±7.1 18.9 ±4.3 3.9 ±5.2 0.53 Synergistic cervical cancer cell line (HeLa) Human drug CAL-DP1 54.9 ±3.9 72.9 ± 2.4 10.7 ±5.5 0.34 Synergistic resistant Colon cancer cell line (HCT-15) Human CAL-DP2 45.25±4.32 51.37±3.67 11.68±2.35 0.59 Synergistic Breast cancer cell line (MDA- MB231) Human CALB-DP 57.37 ±3.46 74.77±4.88 8.25±3.45 0.29 Synergistic Breast cancer cell line (MDA- MB231) CI=(a/A) + (b/B)(ref); CI 1, antagonistic; Chou et al; J. Natl. Cancer Inst. 1994, 86 (20) 1517-1524. EXAMPLE 7: ACTIVE ENDOCYTOSIS OF NANOSPHERENANOCOHLEATE COMPLEXES In vitro cellular uptake study - To study the efficiency of the cellular internalization of the dual delivery nano-complexes the following dye encapsulation schematics were used: CALG-DP 1: Calcein in alginate chitosan nanoparticles and Rhodamine 6G in nanocochleates CALG-DP 2: Rhodamine 6G in alginate-chitosan nanoparticles and Curcumin in nanocochleates CALB- DP: Rhodamine 6G in albumin nanoparticles and Curcumin in nanocochleates All formulations were tested in confluent cancer cells. Briefly, cells were collected from the culture flask and seeded into 24 well plate containing glass coverslip at the density of 5 x 104 cells/well and allowed to adhere overnight. On the next day old media new media containing cochleates (calcein in nanoparticles + rhodamine 6G in lipid bilayer), free calcein (l2mM) into the well and further incubated for 3 h after washing and fixation coverslips were observed under Olympus Fluoview 500 confocal laser-scanning microscope (Olympus, Tokyo, Japan). As shown in Figure 5, Figure 6, Figure 7 both the encapsulated dyes were found internalized within the cancer cell line efficiently. Further, Z scanning of the cell image clearly indicates that the nano-complexes get access to cytoplasm and are not constrained to surface of the cell. This study implies that present invention helps in mediating and facilitating the cellular uptake and delivery of the encapsulated materials. A person skilled in the art will be able to practice the present invention in view of the description presented in this document, which is to be taken as a whole. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. Numerous details and examples have been set forth in order to provide a more thorough understanding of the invention. While the invention has been disclosed in its preferred form, the specific embodiments and examples thereof as disclosed and illustrated herein are not to be considered in a limiting sense. It should be readily apparent to those skilled in the art in view of the present description that the invention can be modified in numerous ways. The inventor regards the subject matter of the invention to include all combinations and sub-combinations of the various elements, features, functions and/or properties disclosed herein. WE CLAIM 1. A nano-complex comprising (i) nanospheres with a first drug entrapped within said nanospheres and (ii) lipid based nanocochleates with a second drug encapsulated therein, characterized in that, said nanospheres are chemically or physically linked either within or on the surface of the nanocochleate such that a combination of drugs are co-encapsulated in a single nanosphere-nanocochleate complex. 2. The nano-complex as claimed in claim 1, wherein the nanospheres are composed of alginate-chitosan, chitosan, gum, albumin, gelatin or collagen. 3. The nano-complex as claimed in claim 1, wherein the nanospheres are biodegradable. 4. The nano-complex as claimed in claim 1, wherein the nanocochleates are composed of phospholipids. 5. The nano-complex as claimed in claim 4, wherein the nanocochleates are composed of phosphotidyl serine [PS], dioleoylphosphatidyl serine [DOPS], phosphatide acid [PA], phosphatidylinositol [PI], phosphatidyl glycerol [PG], phosphatidylcholine [PC], phosphatidylethanolamine [PE], diphosphotidylglycerol [DPG], dioleoyl phosphatidic acid [DOPA], distearoyl phosphatidyl serine [DSPS], dimyristoyl phosphatidyl serine [DMPS], dipalmitoyl phosphatidylglycerol [DPPG] or cholesterol. 6. The nano-complex as claimed in claim 1, wherein the nanocochleate is composed of at least 50% by weight of dioleoylphosphatidylserine 7. The nano-complex as claimed in claim 1, wherein the nano-complex are of 300-850 nm. 8. The nano-co,plex as claimed in 1, wherein the nanosphere is chemically linked on the surface of the nanocochleates using ethylene dicarbiodamide. 9. The nano-complex as claimed in any one of the preceding claims, wherein at least one hydrophobic drug is encapsulated within the nanocochleate and one hydrophilic drug is encapsulated within the nanospheres. 10. The nano-complex as claimed in claim 9, wherein the encapsulation efficiency is at least 75%. 11. The nano-complex as claimed in any one of the preceding claims, wherein is the drug is selected from the group comprising an anti-cancer agent, anti-viral agent, an anesthetic, anti-infectious, steroidal anti-inflammatory, anti-fungal, non-steroidal anti-inflammatory, tranquiliser, nutritional supplement, vasodilatory agent. 12. The nano-complex as claimed in II, wherein the drugs encapsulated are anti-cancer drugs namely paclitaxel and doxorubicin. 13. The nano-complex as claimed in any one of the preceding claims, wherein the anti-cancer drugs exhibits synergistic activity represented by combination index less than 0.9. 14. A process for producing a nano-complex of claim 1 wherein the nanospheres are co-encapsulated within the nanocochleates comprising the steps of (a) A step of preparing drug-contained alginate chitosan nanospheres by means of controlled gelification of alginate in the presence of calcium chloride and chitosan (b) A step of recovery of drug loaded nanoparticles by centrifugation and subsequent washing to obtain chitosan-alginate nanospheres. (c) A step of preparing nanocochleates by dissolving dioleoyl phosphatidyl serine (DOPS) and cholesterol in the ratio of 9:1 in chloroform-methanol. (d) A drug dissolved in methanol is added along with the lipids during the film formation. (e) A thin film of nanocochleates are formed using rotary evaporator at 40°C under vacuum. (f) The film of step (d) is hydrated with the drug contained chitosan-alginate nanospheres in a buffer solution and sonicated. (g) The nanosphere in nanocochleate complex is formed using alginate chitosan nanospheres and purified by centrifugation. 15. A process for producing a nano-complex of claim 1. wherein the nanospheres are linked on the surface of the nanocochleates comprising the steps of (a) A step of preparing drug-contained alginate chitosan nanospheres by means of controlled gelification of alginate in the presence of calcium chloride and chitosan (b) A step of recovery of drug loaded nanoparticles by centrifugation and subsequent washing to obtain chitosan-alginate nanospheres. (c) A step of preparing nanocochleates by dissolving dioleoyl phosphatidylserine (DOPS) and cholesterol in the ratio of 4:1 in chloroform-methanol. (d) A drug dissolved in methanol is added along with the lipids during the film formation. (e) A thin film of nanocochleates are formed using rotary evaporator at 40°C under vacuum. (f) An aliquot of the alginic acid modified chitosan nanoparticle solution is added during the film hydration step. (g) l-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC) and N-hydroxy succinimide (NHS) are added to the cochleate/chhosan-alginate mixture to facilitate covalent bonding between the surface functional groups on nanocochleates and alginate-chitosan nanoparticles. (h) The mixture of step (g) is reacted at room temperature and nanosphere on nanocochleate complexes with alginate chitosan nanospheres were formed and purified with centrifugation. 16. A process of preparing a nano-complex as claimed in claim 15. wherein the nanospheres are composed of albumin prepared by (a) preparing a solution of bovine serum albumin (b) a step of adding ethanol dropwise to the solution of step (a) to facilitate the self assembly of albumin nanoparticles and cross-linking with aqueous 8% glutaraldehyde solution (c) a step of purification by centrifugation, washing and lyophilisation. 17. A pharmaceutical composition comprising the nano-complex of claim 1 and a pharmaceutically effective carrier.

Full Text

FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; rule 13)
TITLE
" A NANOCOCHLEATE-NANOSPHERE COMPLEX "
APPLICANT
Indian Institute of Technology, Bombay, Dept. of Biosciences and Bioengineering, 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 THE INVENTION:
The present invention is directed towards development of nano-complexes comprising nanococleates linked with nanospheres for delivery of two or more drugs. The nanospheres are either chemically or physically bound to the nanocochleates by a simplified procedure which avoids the use of synthetic polymers. Thus, another object of the invention relates to a process for preparing nanocochleate-nanosphere complexes using nanospheres of biodegradable biopolymers and proteins rather than cationic molecules for delivery of hydrophobic and hydrophilic drugs.
BACKGROUND OF THE INVENTION:
Many diseases require therapy with two or more drugs, combinations of the drugs in such a manner as to allow sustained release and reduce the frequency of doses is desirable. One such condition where this would be particularly useful is in cancers. Cancer is the second most common cause of death among adults in most countries. Great progress has been made in the treatment of selected malignancies and approximately 50% of all malignancies can be treated using current treatment strategies. Still, majority of these treatments are carried out by surgery. However, surgical excision is not effective when the cancer cells have infiltrated the nearby vital organs or have spread to other parts of the body. Radiotherapy and chemotherapy used alone or in combination have also shown to greatly improve the management of patients with a variety of solid and haematologic malignancies. Chemotherapeutic drugs may destroy healthy tissue along with the cancer cells and also give rise to adverse side effects such as fatigue, vomiting, alopecia, gastro-intestinal disturbances, anemia etc. Further, by oral delivery of the chemotherapeutic agents about 8-10 of the active drug is either denatured by the stomach environment or eliminated via liver metabolism. Thus, despite improvements in the treatment of most metastatic solid tumors, these remain largely incurable. Reasons for this are insufficient tumor selectivity of anti-cancer agents and poor penetration within the tumor mass.

To overcome the failure of conventional therapy, combination of two different cytotoxic chemotherapeutic agents is given. This combination therapy improves cancer remission rate and duration of survival. At present, combination therapy is achieved by two or more drugs being administered separately which does not allow one to achieve controlled and sustained release from the formulations. In addition, there is a need to co-encapsulate hydrophobic and hydrophilic drugs in the same carrier through non-invasive routes like oral or aerosol.
In this domain, particularly for dual delivery of drugs, nanotechnology based therapeutics have given rise to enormous potential in addressing the failures of traditional therapeutics that could not be effectively formulated owing to poor water solubility or lack of target specificity. Nanoparticles are preferred for their properties such as biodegradability, biocompatibility, conjugation, encapsulation and their ability to be functionalized. Further, these nanoparticles are superior over conventional drug delivery methods as these can be targeted to a specific area and owing to their extremely small size they can penetrate through smaller capillaries and are easily taken up by cancer cells.
US 7,304,045 B2 describes a dual particle tumor targeting system consisting of a galactosamine conjugated nanoparticle and a second EPR mediated nanoparticle mixed together in a solution. Also, the two nanoparticles are simply administered at different time points as there is no single composite entity formed by the interaction of the nanoparticles There is no covalent interaction between the two nanostructures, thereby making the targeting of the second nanoparticle passive. On the other hand, the present invention does not require administration of the two particles at different time points as the constituent nanospheres and nanocochleates are engineered to release the drugs at different time points.
Patent No. US 7,244,781 B2 describes the method of preparation of composite compositions including polymeric nanoparticles and clay nanoparticles. The nanoparticles, being non biodegradable and non-biocompatible, would be cleared by the reticulo-endothelial system and elicit immune response by the body.

US 2010/0203163 describes an injectable polymer-lipid blend provided as a localized drug delivery system for a pharmaceutically active agent. The effectiveness of the system is dependent upon the rheological properties and the viscoelasticity of the blend. Also, it is distinct from the dual-delivery system developed by virtue of being a matrix based system.
US 7511023 and US 2004/0138152 describes the method of preparation of anticancer drug-chitosan complex forming self aggregates in aqueous media composed of a hydrophobic anticancer agent and a hydrophilic chitosan. The present invention deals with alginate-chitosan nanoparticles complexed with lipid nanocochleates for dual drug delivery.
US 7,740,883 and US 2005/0226938 describe methods for preparing cross linked core and core-shell nanoparticle polymers from chitosan. The dual delivery system developed by us comprises of chitosan nanoparticles specifically modified with alginic acid to increase the encapsulation of hydrophilic anthracycline drugs.
US 2009/0036417 and US 2004/0092727 describe cochleate and nanocochleate systems wherein the agents bridging agents are organic multi-valent cations. In the dual-delivery system developed by us, nanoparticles are used as bridging agents for formation of cochleates.
US 20030185894 describes a process for producing nanoparticles of hydrophobic drug paclitaxel and albumin. In the dual delivery nanocomplex system developed by us, albumin nanoparticles are used to encapsulate the hydrophilic drug doxorubicin.
US20060246524 and US20090181398 describe conjugate compositions that include a specific-binding moiety covalently coupled to a nanoparticle through a heterobifunctional polyalkyleneglycol linker. In the dual nano-complex system developed by us, two distinct species of nanoparticles are physically or chemically bound together without the use of any linker molecule.

US 2009/0053297 describes negatively charged and PEGylated lipid structures (liposomes, cochleates, micelles) for reducing the immunogenicity and increasing the half life of therapeutic proteins such as Factor VIII. The system uses a single lipid structure to encapsulate therapeutic proteins. It does not deal with nanosphere-nanocochleate complexes for combination drug delivery.
US Patents 4078052 and 5840707 deal with the development of cochleates from large unilamellar vesicles and for the delivery of proteins or peptides as vaccines. These patents do not deal with control of sizes in the form of nanocochleates and do not deal with nanocomplexes for dual drug delivery.
US Patent 6153217 deals with a process for the preparation of nanocochleates for oral or mucosal drug delivery. It comprises of preparing small unilamellar liposomes of negatively charged lipid mainly phosphatidylserine and mixing the liposome suspension with synthetic polymer A consisting of dextran or polyethyleneglycol 2-20%w/w. The liposome/polymer suspension is then added into a solution of polymer B (wherein polymers A and B are immiscible) like polyvinylpyrolidone or polyvinylalcohol or Ficoll or polyvinylmethylether or polyethylene glycol 2-20%w/w. Finally a solution of a positively charged molecule which is a cationic salt is added to the two phase polymer system and excess polymer is removed by centrifugation.
Despite the aforesaid, there is still a need for a unique nano-complex system comprising two different nanoparticle systems with better physiochemical properties, anti-tumor activity and targeting ability. Furthermore, the present invention deals with nanosphere-nanocochleate complexes in which the nanocochleates are held together by biopolymeric or protein nanospheres instead of adding cations or drugs (as used in prior art) to facilitate the acquisition of their characteristic multilayered spiral structure.
Thus, the present invention deals with the development of nanocochleate-nanosphere complexes with a new process where biopolymeric and protein nanospheres are formed and directly added during the hydration of the unilamellar liposomes to form nanocochleate-

nanosphere complexes for dual drug delivery (both hydrophobic and hydrophilic in nature). The invention employs alginate-chitosan and albumin nanospheres for causing precipitation of the nano-cochleate-nanosphere complexes for dual drug delivery.
SUMMARY OF THE INVENTION
An object of this invention is to develop biodegradable lipopolymeric and lipoprotein nanocochleate-nanosphere complexes for delivery of both hydrophobic and hydrophilic drugs.
Another object is to provide sustained release, gastric resistance and synergistic action of the dual drugs encapsulated within the nanocomplexes.
Another object of the invention is to provide active targeting and endocytosis of the nanocochleate-nanosphere complex.
A further object of this invention is in process for preparing nanocohleate-nanosphere complexes using nanospheres of biodegradable biopolymers and proteins rather than cationic molecules, for delivery hydrophobic and hydrophilic drugs.
ADVANTAGES
The present invention has the following advantages over the prior art:
Biodegradable biopolymeric and protein nanospheres stably linked with lipid nanocochleates
- simple process for preparation which avoids cationic salts for crosslinking
- size of 300-800 nm
- shows high cellular internalization by endocytosis in cancer cells
- stable in harsh conditions of gastric acids and enzymes
delivers hydrophobic and hydrophilic drugs in a sustained manner

Other features and advantages of the present invention will become apparent as the following detailed description proceeds or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing summary, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:
Figure 1: A & B- Transmission electron microscopic image of chitosan alginate
nanospheres; C & D- Transmission electron microscopic image of nanosphere on
nanocochleate complex using chitosan alginate nanospheres
Figure 2: A & B- Transmission electron microscopic image of nanosphere in
nanocochleate complex using chitosan alginate nanospheres.
Figure 3: A & B- Transmission electron microscopic image of albumin nanospheres; C &
D- Transmission electron microscopic image of nanosphere on nanocochleate complex
using albumin nanospheres
Figure 4: Confocal Microscopy images of Calcein encapsulated Alginate chitosan
nanospheres encochleated inside nanocochleates containing Rhodamine 6G dye (5µm)
(without any drugs)
Figure 5: Cellular internalization of the calcein loaded alginate chitosan nanospheres
(green) B with rhodamine 6G nanocochleates (red) A in HCT-15 cancer cell line by
confocal laser scanning microscopy (both the images were captured at the same location
with two different laser source) without any drugs

Figure 6: Cellular internalization of the Rhodamine-6G loaded albumin nanospheres (red) B with curcumin loaded nanocochleates (green) A in MDA-MB 231 cancer cell line by confocal laser scanning microscopy (both the images were captured at the same location with two different laser source) (without any drugs)
Figure 7: Cellular internalization of the Rhodamine-6G loaded alginate chitosan nanospheres (red) B with curcumin loaded nanocochleates (green) A in MDA-MB 231 cancer cell line by confocal laser scanning microscopy (both the images were captured at the same location with two different laser source) (without any drugs) Figure 8: A- Paclitaxel release from nanosphere in nanocochleates complex using alginate chitosan nanospheres versus marketed standard paclitaxel formulation; B Doxorubicin release from same nanosphere- nanocochleates complex versus standard doxorubicin Figure 9: A- Paclitaxel release from nanosphere on nanocochleate complexes using alginate chitosan nanospheres versus marketed standard paclitaxel formulation; B Doxorubicin release from nanosphere-nanocochleate complex versus standard doxorubicin Figure 10: A- Paclitaxel release from nanosphere on nanocochleate complex using albumin nanospheres versus marketed standard paclitaxel formulation; B-Doxorubicin release from same nanosphere nanocochleates complex formulation versus standard doxorubicin
Figure 11: Drug release in simulated gastric fluid(0.1 M Hydrochloric acid) at two different intervals
DETAILED DESCRIPTION OF THE INVENTION
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section. Specific and preferred values listed below for individual components, substituents,

and ranges are for illustration only; they do not exclude other defined values or other values within defined ranges for the components and substituents.
As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified systems or process parameters that may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the scope of the invention in any manner.
In our present invention we have encapsulated two different drugs for combination chemotherapy in two different nanoparticle systems which are combined together to forma unique nano-complex system with better physiochemical properties, anti-tumour activity and targeting ability than either of the constituents. In these regard spherical nanoparticles were prepared from biopolymers and their derivatives (such as chitosan, alginate, albumin) encapsulating drug 1(D1). These nanoparticles were used as complexing agent for the preparation of nanocochleates made up of lipids such as Phosphatidylserine (DOPS), Phosphatidylcholine (DPPC) and Cholesterol.
The invention deals with nanosphere-nanocochleate complexes in which the nanocochleates are held together by biopolymeric or protein nanospheres instead of adding cations or drugs (as used in prior art) to facilitate the acquisition of their characteristic multilayered spiral structure. The anticancer drug 1 (Dl) entrapped (or adsorbed on surface) within spherical nanoparticles helps in the formation of nanocochleate like structure while the drug (D2) is encapsulated within the bilayer of the nanocochleates. Two distinct species of nano-complexes were obtained by this method depending upon the size of the spherical nanoparticles: (i) Nanosphere in nanocochleate: Spherical nanoparticles entrapped inside spiral nanocochleates for (ii) Nanosphere on Nanocohleate: Spherical nanoparticles covalently attached on the surface of the spiral nanocochleates.

In context of the present invention, nanocochleates are cylindrical (cigar-like) microstructures that consist of a series of lipid bilayers which are formed as a result of the condensation of small unilamellar negatively charged liposomes. They have a unique multilayered structure consisting of a solid, lipid bilayer sheet rolled up in a spiral or in stacked sheets, with little or no internal aqueous space. Because the entire nanocochleate structure is a series of solid layers, components encapsulate within the interior of the nanocohleate structure and remain intact, even though the outer layers of the nanocochleate may be exposed to harsh environmental conditions. The drug is preferably encapsulated in the lipid bilayer of the nanocochleates.
The different lipids that make up the nanocochleates include but are not limited to phosphotidyl serine [PS], dioleoylphosphatidyl serine [DOPS], phosphatidic acid [PA], phosphatidylinositol [PI], phosphatidyl glycerol [PG], phosphatidylcholine [PC], phosphatidylethanolamine [PE], diphosphoticlylglycerol [DPG], dioleoyl phosphatidic acid [DOPA], distearoyl phosphatidyl serine [DSPS], dimyristoyl phosphatidyl serine [DMPS], dipalmitoyl phosphatidylglycerol [DPPG] or cholesterol. Nanocochleates of the present invention, without intending to be limited therein are preferably made of the lipid dioleyl phosphatidylserine [DOPS].
According to a preferred embodiment of the present invention, the lipid Dioleyl phosphatidylserine (DOPS) was employed as major component for formation of the structure. DOPS is an anionic phospholipid which tends to provide negative charge so as to facilitate the assembly of the spiral multilayered structure. In addition, DOPS is a completely biocompatible and biodegradable lipid. It is, in fact, of nutritious value and has benefits in memory and learning enhancement. In addition to it cholesterol and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine) were added to improve the stability of the structure during storage as well as when administered for activity.
The particle size of the nanocochleates are preferably in the range of 300 - 500 nm.
In accordance to the instant invention, nanospheres are solid-state nanoparticles ranging from 10 to 200 nm in size. These are mainly spherical particles which can be either

amorphous or crystalline in nature and have the ability to protect the drug from enzymatic and chemical degradation. The drug is dissolved or encapsulated or attached to the nanoparticles and, depending on the methods employed for its preparation; one can obtain nanospheres, nanocapsules or aquasomes. Nanospheres are generally composed of natural polymers such as chitosan, gum, albumin, gelatin or collagen and the drug is uniformly dispersed in it. The tiny capsule of drug store house is called vesicle and the solid skeleton structure is called nanosphere. The nanospheres of the present invention are biodegradable in nature.
The particle size of the nanospheres are preferably in the range of 40 - 100 nm.
Nano-complexes meant for dual delivery of pharmaceutically active entities in accordance to the present invention comprises nanospheres linked with nanocochleates wherein the nanosphere is either entrapped inside the nanocochleate or covalently attached on the surface of the nanocochleate. Thus, nano-complexes meant for dual delivery of pharmaceutically active entities consisting of lipid nanocochleates (particle size 300-500 nm) either incorporating or covalently attaching comparatively smaller spherical nanoparticles on their surfaces (particle size 40-100 nm). The active entities are encapsulated into the nanocochleates and the nanospheres, thereby ensuring simultaneous delivery of both the pharmaceuticals for combination chemotherapy. Doxorubicin and paclitaxel were the drugs used as the representative hydrophobic and hydrophilic drugs encapsulated within the nano-complexes. This formulation prepared intended for different routes of administration from oral to aerosol. Therefore, the present developed formulation comprising two different anticancer drugs tend to show good anticancer activity than when it is administered separately in multidrug resistant cells.
One aspect of the invention relates to a nano-complex comprising nanospheres with a first drug entrapped within said nanospheres and lipid based nanocochleates with a second drug encapsulated therein, characterized in that, said nanospheres are chemically or physically linked either within or on the surface of the nanocochleate such that a combination of drugs are co-encapsulated in a single nanosphere-nanocochleate complex.

The present invention however contemplates the use of two or more drugs for encapsulation and delivery.
More specifically, the present invention is a nano-complex delivery vehicle comprising of a nanocochleate formulation associated with smaller spherical nanoparticles, which in turn, also serve as complexing agent for nanocochleate formation. These nanostructures comprise of two different anti-cancer drugs for effective treatment of the cancer by combination chemotherapy. The spherical nanoparticles (made up of biopolymers such as alginate, chitosan, albumin) contain the hydrophilic anticancer drug and the other hydrophobic anticancer drug is encapsulated in the lipid bilayer of the nanocochleates. Thus two different anticancer drugs are co-encapsulated in one single nano-complex thereby facilitating synergistic anticancer activity.
The present invention although explains the use of nano-complexes for dual delivery of drugs for combination chemotherapy but the scope is not restricted to anticancer agents per se. The drug may be an anti-cancer agent, anti-viral agent , an anesthetic, anti-infectious, steroidal anti-inflammatory, anti-fungal, non-steroidal anti-inflammatory, tranquiliser, nutritional supplement, vasodilatory agent and the like. These nanostructures also help various other pharmaceutically active entities to cross cell barriers, permit solubilization and protects compounds during transport owing to their unique physiochemical properties.
EXAMPLES
The following examples are provided to better illustrate the claimed invention and are not to be interpreted in any way as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in

the procedures herein described while still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.
EXAMPLES
EXAMPLE 1: PREPARATION OF NANOSPHERE IN NANOCOCHLEATE COMPLEXES USING ALGINATE CHITOSAN NANOSPHERES PREPARATION OF ALGINATE CHITOSAN NANOSPHERES (CALG-1)
Alginate chitosan nanoparticles were prepared using controlled gelification of alginate in presence of cation and chitosan. Briefly, calcium chloride (0.5 ml, 18mM) was added to 9.5 ml of sodium alginate solution (0.06% w/v) containing drug. 2 ml of chitosan solution (0.05% w/v) was added followed by stirring for 30 min and the mixture was kept overnight at room temperature. Drug loaded nanoparticles were recovered by centrifugation at 32000 rpm for 30-45 min and washed thrice with TES buffer to obtain the final pellet. The supernatant solution was used to determine the concentration of unentrapped drug (doxorubicin (Dl)'by using fluorescence spectrometer with excitation and emission wavelength of 470nm and 580 nm.
PREPARATION OF NANOCOCHLEATES ENCAPSULATING ALGINATE-CHITOSAN NANOSPHERES (NANOSPHERE-IN NANOCOHLEATE) (CAL-DP 1)
Dioleyl Phosphatidylserine (DOPS) and cholesterol in the ratio of 9:1 were dissolved in chloroform-methanol (2:1). Thin film of lipids was formed using rotary evaporator at 40°C under vacuum. For drug-D2 (paclitaxel) encapsulation, drug dissolved in methanol and added along with lipids during film formation step. Then film was hydrated with nanoparticles in TES-Buffer to get lipid concentration of 10mg lipid/ml at 45°C for 20 min and sonicated for a minute using probe sonicator. Thus encochleated nanoparticles centrifuged at 25,000g for 10 min to separate un-encapsulated nanoparticles. The pellet reconstituted accordingly with TES Buffer containing 0.5mM of calcium chloride.

EXAMPLE 2: PREPARATION OF NANOSPHERE ON NANOCOCHLEATE COMPLEXES USING ALGINATE_CHITOSAN NANOSPHERES
2.1. PREPARATION OF ALGINATE-CHITOSAN NANOPARTICLES AS DESCRIBED IN EXAMPLE 1
2.2. PREPARATION OF NANOCOCHLEATES WITH COVALENTLY ATTACHED ALGINATE-CHITOSAN
NANOPARTICLES (CAL-DP2)
For the preparation of the dual delivery nano-complex, a cochleate suspension comprising the lipids DOPS and DPPC (in the ratio 4:1) and encapsulating drug D2 (paclitaxel) was prepared by the method stated above in example 1(b). The aliquot of the alginic acid modified chitosan nanoparticle solution was added during the film hydration step. 100mg l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and 50mg N-hydroxysuccinimide (NHS) were also added to the cochleate/chitosan-alginate mixture to facilitate covalent bonding between the surface functional groups of nanocochleates and alginate-chitosan nanoparticles. The carbodiimide was solubilized using a vortex mixture. The mixture was reacted for 2h at room temperature (6) and was centrifuged. The supernatant of the centrifuged solution was discarded and the pellet was re-suspended in 10 ml of TES buffer. The resultant suspension was then sonicated and then lyophilized.
EXAMPLE 3: PREPARATION OF NANOSPHERE ON NANOCOCHLEATE
COMPLEXES USING ALBUMIN NANOSPHERES
3.1. PREPARATION OF ALBUMIN NANOPARTICLES (ALB)
For the preparation of albumin nanoparticles, aliquots of 4.0 mg of doxorubicin and 20 mg of Bovine Serum Albumin (BSA) were dissolved in 1ml of purified water and stirred for 2 h. 3ml of ethanol was added drop wise to facilitate the self assembly of albumin nanoparticles. Further it was crosslinked using aqueous 8% glutaraldehyde solution (0.294-1.175µl/mg BSA, 24 h). The pH was adjusted to 8.2 after carrying out the desolvation step at pH 6.5. Further particles were purified by centrifugation at 25000g for 20 minutes, and then dispersed in water and lyophilized. The supernatant solution was used to determine the concentration of un-entrapped doxorubicin (Dl) by using fluorescence spectrometer with excitation and emission wavelength of 480nm and 550 nm.

3.2. PREPARATION OF NANOCOCHLEATES WITH COVALENTLY ATTACHED ALBUMIN NANOPARTICLES (CALB-DP)
For the preparation of the dual delivery nano-complex, a cochleate suspension comprising the lipids DOPS and DPPC (in the ratio 4:1) and encapsulating drug D2 (paclitaxel) was prepared by the method stated above in example 1(b). The aliquot of the albumin nanoparticle solution was added during the film hydration step. 100mg EDC and 50mg N-hydroxy succinimide (NHS) were also added to the cochleate/albumin mixture to facilitate covalent bonding between the surface functional groups of nanocochleates and albumin nanoparticles. The carbodiimide was solubilized using a vortex mixture. The mixture was reacted for 2h at room temperature and was centrifuged. The supernatant of the centrifuged solution was discarded and the pellet was re-suspended in 10 ml of TES buffer. The resultant suspension was then sonicated. The resultant suspension was lyophilized and FT-IR spectroscopy was carried out.
EXAMPLE 4
Nanosphere-nanocochleate systems as described in example 1 wherein the method of alginate-chitosan nanosphere formation is slightly modified: About 1% chitosan solution was prepared in 1% acetic acid. 1% TPP solution was prepared in water and an equivalent amount of alginic acid was added to it and the mixture was incubated overnight in the dark. Nanoparticles were spontaneously obtained under constant stirring at room temperature for 1 hour at 1000 rpm. The prepared nanoparticles were separated by centrifugation at 25000g for 20 minutes, and then dispersed in water and lyophilized, followed by steps for incorporation in nanocochleates as described in example 1.
EXAMPLE 5:
5.1. CHARACTERISATION OF NANOSPHERE NANOCOCHLEATE
COMPLEXES
The typical structure of the nanoparticles and nanocochleates was confirmed by using electron microscopy (Transmission and scanning electron microscopy). Particle size

measured using dynamic light scattering (Table 1) were in coherence with particles size as observed in electron microscopy studies (Figure 2, Figure 1, Figure 1). The Zeta potential of the different formulations is stated in Table 1.
TABLE 1: PHYSIOCHEMICAL PROPERTIES OF DIFFERENT FORMULATIONS:

Formulation Particle size(nm) Polydispersity index Zeta potential (mV)
Alginate chitosan 32.7 ±4.6 0.14 ±0.29 -14.7 ±6.9
nanospheres
Nanosphere in 482.3 ±223.1 0.32 ±0.18 -72.8 ±11.4
nanocochleate using
chitosan alginate
nanospheres

Nanosphere on 836.6 ±199.1 0.45 ±0.12 -20.9 ±1.5
nanocochleate using
chitosan alginate
nanosphere
Albumin nanospheres 280.3 ± 8.2 0.14± 0.31 -16±2.6
Nanosphere on 519.4 ±45.2 0.21 ±0.20 -31 ±3.5
nanocochleates using
albumin nanospheres
Further to confirm the nanoparticle in cochleate. confocal microscopy of the same was carried out with two different dyes (cresol red and calcein). Rhodamine 6G (lmg/ml) was dissolved in ethanol and added along with lipids during thin film formation. On the other hand Calcein loaded nanoparticles were prepared separately and nanocochleates were prepared out of them as explained in example 1,2,3.The confocal microscopy images confirmed the formation of needle shaped cochleate like structure (Figure 4) with two different dyes.

5.2. ENCAPSULATION AND IN VITRO RELEASE OF DRUGS FROM NANOSPHERE-NANOCOCHLEATECOMPLEXES
The invented formulation showed high encapsulation of paclitaxel (Table 2). Similarly, high entrapment of hydrophilic drug such as doxorubicin was observed (Table 2).
TABLE 2: ENCAPSULATION EFFICIENCY OF DRUGS

Encapsulation efficiency (%)
Drug Paclitaxel Doxorubicin
CAL-DP-1 81.9±7.7 66.2 ±2.2
CAL-DP-2 76.8 ±4.3 73.8 ±0.3
CALB-DP 79.3 ±6.1 89.2 ± 0.4
In vitro release was performed for two different drugs using dialysis bag method in 25% methanolic phosphate buffer (pH 7.4). Methanol was incorporated in the media in order to overcome the solubility issue of the drug.
5.2.1. CAL-DP1: Approximately 30% paclitaxel was released from cochleates into release media over 48 h of dialysis where as standard PTX released 85% with in 3h shows sustained and controlled release of drug from the cochleates (Figure 8). Similarly, doxorubicin as estimated by fluorescence spectrometer (R2 = 0.989) was found to be released 76% of the total drug in 24h in comparison to the free standard doxorubicin released similar quantity in6 0 min explains the controlling release nature of the two drugs from the formulation (Figure 8). To test the cochleate formulation for oral delivery we tried to identify the gastric resistance of the same in 25% methanolic 0.1M hydrochloric acid (pH 1.2) by in vitro release using dialysis bag method for an hour. Paclitaxel and Doxorubicin released only 10% and 37% from the cochleate respectively and found to exhibit gastric resistance (Figure 11). Thus this formulation can also be administered via oral route.

5.2.2. CAL-DP2: The percentage of Paclitaxel released from cochleates was about 22% in
26 hours (Figure 9). Doxorubicin as estimated by fluorescence spectrometer (R2 = 0.989)
was found to be released 60% in 26h (Figure 9). There was a marked
improvement in the control of release of Doxorubicin from chitosan alginate nanoparticles covalently attached to nanocochleates as compared to the free standard doxorubicin. This formulation can also be administered via oral route owing to its controlled release and gastric resistance as shown by the reduction in release of both paclitaxel and doxorubicin at acidic pH (Figure 11).
5.2.3. CALB-DP: The percentage of Paclitaxel released from cochleates was about 19% in
72 hours (Figure 10). Doxorubicin as estimated by fluorescence spectrometer (R2 = 0.989)
was found to be released 60% in 72h (Figure 10). There was a marked improvement in the
control of release of Doxorubicin from albumin nanoparticles covalently attached to
nanocochleates as compared to the free standard doxorubicin. This formulation can be
considered for intravenous delivery. Oral administration is also possible owing to the
presence of specific uptake pathways for albumin, a natural nutrient for the body
EXAMPLE 6: SYNERGISTIC ACTION OF DUAL DRUGS LOADED WITHIN NANOSPHERE_NANOCOHLEATE COMPLEXES
In vitro anti-cancer activity - The formulations were tested for cytotoxicity in the following cell lines:
CAL-DP 1: HeLa (drug sensitive cell line), HCT-15(multi drug resistant cancer cell line) CAL-DP 2 and CALB-DP: MDA-MB 231 (multidrug resistant cell line) The IC-50 values are shown in the Table 3. As compared to PTX or DOX alone, the combination index calculated as explained by chou et al showed synergistic activity. Thus two drug combination within the cochleate showed synergistic effect in killing the multi¬drug resistant cancer cell line.
TABLE 3: INHIBITORY CONCENTRATION (IC50 nM) OF ANTICANCER (PACLITAXEL) FORMULATION ON DIFFERENT CANCER CELL LINES (n=3)

IC50 (nM)
Cancer cell Formulation PTX DOX PTX+DOX Combination Interaction
line index type
Human CAL-DP1 12±7.1 18.9 ±4.3 3.9 ±5.2 0.53 Synergistic
cervical
cancer cell
line (HeLa)
Human drug CAL-DP1 54.9 ±3.9 72.9 ± 2.4 10.7 ±5.5 0.34 Synergistic
resistant
Colon cancer
cell line
(HCT-15)
Human CAL-DP2 45.25±4.32 51.37±3.67 11.68±2.35 0.59 Synergistic
Breast cancer
cell line
(MDA-
MB231)
Human CALB-DP 57.37 ±3.46 74.77±4.88 8.25±3.45 0.29 Synergistic
Breast cancer
cell line
(MDA-
MB231)
CI=(a/A) + (b/B)(ref); CI 1, antagonistic; Chou et al; J.
Natl. Cancer Inst. 1994, 86 (20) 1517-1524.
EXAMPLE 7: ACTIVE ENDOCYTOSIS OF NANOSPHERENANOCOHLEATE COMPLEXES
In vitro cellular uptake study - To study the efficiency of the cellular internalization of the dual delivery nano-complexes the following dye encapsulation schematics were used: CALG-DP 1: Calcein in alginate chitosan nanoparticles and Rhodamine 6G in nanocochleates

CALG-DP 2: Rhodamine 6G in alginate-chitosan nanoparticles and Curcumin in nanocochleates
CALB- DP: Rhodamine 6G in albumin nanoparticles and Curcumin in nanocochleates All formulations were tested in confluent cancer cells. Briefly, cells were collected from the culture flask and seeded into 24 well plate containing glass coverslip at the density of 5 x 104 cells/well and allowed to adhere overnight. On the next day old media new media containing cochleates (calcein in nanoparticles + rhodamine 6G in lipid bilayer), free calcein (l2mM) into the well and further incubated for 3 h after washing and fixation coverslips were observed under Olympus Fluoview 500 confocal laser-scanning microscope (Olympus, Tokyo, Japan). As shown in Figure 5, Figure 6, Figure 7 both the encapsulated dyes were found internalized within the cancer cell line efficiently. Further, Z scanning of the cell image clearly indicates that the nano-complexes get access to cytoplasm and are not constrained to surface of the cell. This study implies that present invention helps in mediating and facilitating the cellular uptake and delivery of the encapsulated materials.
A person skilled in the art will be able to practice the present invention in view of the description presented in this document, which is to be taken as a whole. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. Numerous details and examples have been set forth in order to provide a more thorough understanding of the invention. While the invention has been disclosed in its preferred form, the specific embodiments and examples thereof as disclosed and illustrated herein are not to be considered in a limiting sense. It should be readily apparent to those skilled in the art in view of the present description that the invention can be modified in numerous ways. The inventor regards the subject matter of the invention to include all combinations and sub-combinations of the various elements, features, functions and/or properties disclosed herein.

WE CLAIM
1. A nano-complex comprising (i) nanospheres with a first drug entrapped within said nanospheres and (ii) lipid based nanocochleates with a second drug encapsulated therein, characterized in that, said nanospheres are chemically or physically linked either within or on the surface of the nanocochleate such that a combination of drugs are co-encapsulated in a single nanosphere-nanocochleate complex.
2. The nano-complex as claimed in claim 1, wherein the nanospheres are composed of alginate-chitosan, chitosan, gum, albumin, gelatin or collagen.
3. The nano-complex as claimed in claim 1, wherein the nanospheres are biodegradable.
4. The nano-complex as claimed in claim 1, wherein the nanocochleates are composed of phospholipids.
5. The nano-complex as claimed in claim 4, wherein the nanocochleates are composed of phosphotidyl serine [PS], dioleoylphosphatidyl serine [DOPS], phosphatide acid [PA], phosphatidylinositol [PI], phosphatidyl glycerol [PG], phosphatidylcholine [PC], phosphatidylethanolamine [PE], diphosphotidylglycerol [DPG], dioleoyl phosphatidic acid [DOPA], distearoyl phosphatidyl serine [DSPS], dimyristoyl phosphatidyl serine [DMPS], dipalmitoyl phosphatidylglycerol [DPPG] or cholesterol.
6. The nano-complex as claimed in claim 1, wherein the nanocochleate is composed of at least 50% by weight of dioleoylphosphatidylserine
7. The nano-complex as claimed in claim 1, wherein the nano-complex are of 300-850 nm.
8. The nano-co,plex as claimed in 1, wherein the nanosphere is chemically linked on the surface of the nanocochleates using ethylene dicarbiodamide.
9. The nano-complex as claimed in any one of the preceding claims, wherein at least one hydrophobic drug is encapsulated within the nanocochleate and one hydrophilic drug is encapsulated within the nanospheres.

10. The nano-complex as claimed in claim 9, wherein the encapsulation efficiency is at least 75%.
11. The nano-complex as claimed in any one of the preceding claims, wherein is the drug is selected from the group comprising an anti-cancer agent, anti-viral agent, an anesthetic, anti-infectious, steroidal anti-inflammatory, anti-fungal, non-steroidal anti-inflammatory, tranquiliser, nutritional supplement, vasodilatory agent.
12. The nano-complex as claimed in II, wherein the drugs encapsulated are anti-cancer drugs namely paclitaxel and doxorubicin.
13. The nano-complex as claimed in any one of the preceding claims, wherein the anti-cancer drugs exhibits synergistic activity represented by combination index less than 0.9.
14. A process for producing a nano-complex of claim 1 wherein the nanospheres are co-encapsulated within the nanocochleates comprising the steps of

(a) A step of preparing drug-contained alginate chitosan nanospheres by means of controlled gelification of alginate in the presence of calcium chloride and chitosan
(b) A step of recovery of drug loaded nanoparticles by centrifugation and subsequent washing to obtain chitosan-alginate nanospheres.
(c) A step of preparing nanocochleates by dissolving dioleoyl phosphatidyl serine (DOPS) and cholesterol in the ratio of 9:1 in chloroform-methanol.
(d) A drug dissolved in methanol is added along with the lipids during the film formation.
(e) A thin film of nanocochleates are formed using rotary evaporator at 40°C under vacuum.
(f) The film of step (d) is hydrated with the drug contained chitosan-alginate nanospheres in a buffer solution and sonicated.
(g) The nanosphere in nanocochleate complex is formed using alginate chitosan nanospheres and purified by centrifugation.

15. A process for producing a nano-complex of claim 1. wherein the nanospheres are
linked on the surface of the nanocochleates comprising the steps of
(a) A step of preparing drug-contained alginate chitosan nanospheres by means of controlled gelification of alginate in the presence of calcium chloride and chitosan
(b) A step of recovery of drug loaded nanoparticles by centrifugation and subsequent washing to obtain chitosan-alginate nanospheres.
(c) A step of preparing nanocochleates by dissolving dioleoyl phosphatidylserine (DOPS) and cholesterol in the ratio of 4:1 in chloroform-methanol.
(d) A drug dissolved in methanol is added along with the lipids during the film formation.
(e) A thin film of nanocochleates are formed using rotary evaporator at 40°C under vacuum.
(f) An aliquot of the alginic acid modified chitosan nanoparticle solution is added during the film hydration step.
(g) l-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC) and N-hydroxy succinimide (NHS) are added to the cochleate/chhosan-alginate mixture to facilitate covalent bonding between the surface functional groups on nanocochleates and alginate-chitosan nanoparticles.
(h) The mixture of step (g) is reacted at room temperature and nanosphere on
nanocochleate complexes with alginate chitosan nanospheres were formed and purified with centrifugation.
16. A process of preparing a nano-complex as claimed in claim 15. wherein the
nanospheres are composed of albumin prepared by (a) preparing a solution of
bovine serum albumin (b) a step of adding ethanol dropwise to the solution of step
(a) to facilitate the self assembly of albumin nanoparticles and cross-linking with

aqueous 8% glutaraldehyde solution (c) a step of purification by centrifugation, washing and lyophilisation.
17. A pharmaceutical composition comprising the nano-complex of claim 1 and a pharmaceutically effective carrier.

Documents:

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Patent Number

272767

Indian Patent Application Number

2190/MUM/2011

PG Journal Number

18/2016

Publication Date

29-Apr-2016

Grant Date

26-Apr-2016

Date of Filing

02-Aug-2011

Name of Patentee

INDIAN INSTITUTE OF TECHNOLOGY, BOMBAY

Applicant Address

DEPT.OF BIOSCIENCES AND BIOENGINEERING,POWAI,MUMBAI-400076,MAHARASHTRA,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	PROF. RINTI BANERJEE	INDIAN INSTITUTE OF TECHNOLOGY,BOMBAY, BIOSCIENCES AND BIOENGINEERING,POWAI,MUMBAI-400076,MAHARASHTRA,INDIA
2	THANIGAIVEL S	INDIAN INSTITUTE OF TECHNOLOGY,BOMBAY, BIOSCIENCES AND BIOENGINEERING,POWAI,MUMBAI-400076,MAHARASHTRA,INDIA
3	SUDEEP CHAKRAVARTY	INDIAN INSTITUTE OF TECHNOLOGY,BOMBAY, BIOSCIENCES AND BIOENGINEERING,POWAI,MUMBAI-400076,MAHARASHTRA,INDIA

PCT International Classification Number

A61K 38/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA