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
"CONJOINED LIPID BASED MULTI COMPARTMENT NANOVESICLES
FOR DRUG DELIVERY "
APPLICANT
Indian Institute of Technology, Bombay, Department of Biosciences and Bioengineering, Powai, Mumbai-400 076 Maharashtra India
The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF INVENTION
The present invention relates to a novel two compartment nanovesicles that can deliver two or more anticancer drugs for synergistic action in response to multiple triggers like pH, temperature and enzymes.
BACKGROUND OF THE INVENTION
Cancer results from aberrant cell cycle progression, leading to enormous proliferation of cells. Current treatment approaches include surgery, chemotherapy, radiation therapy etc. As far as conventional chemotherapy is considered, it is characterized by the "Hill and Valley" phenomena. As the drug is administered, plasma drug concentration increases, peaks and then drops as the drug is metabolized. This cycle is repeated for each drug dose. This is not desirable as the "peak" concentrations of the drug could be toxic to normal cells and the "trough" levels could be ineffective. Thus the therapeutic concentration of the drug is reached only intermittently. For efficient treatment, sustained release of drugs that can maintain the plasma concentrations of the drug to be in therapeutic window for a prolonged period of time are desired.
Chemotherapeutic treatment of tumors is limited by the low therapeutic index of the anticancer drugs currently used. The reasons for this being low anticancer activity accompanied with severe side effects. Also the drugs accumulate not only in tumors but also in healthy tissues, which often results in no tumor targeting at all. Anticancer drugs like paclitaxel are highly hydrophobic, and hence are not soluble in aqueous medium and relevant pharmaceutical solvents. Clinical

administration of many such anticancer drugs requires adjuvants, which may involve numerous side affects and sometimes could be fatal. Currently, combination chemotherapy is given by using free drugs separately with no control of the release of the individual drugs. A solution was searched for the aforementioned problems.
Prior art related to nanovesicle systems for the delivery of multiple anticancer drugs is limited.
US patent 7,850,990, reports a liposome capable of co-encapsulating two different
anticancer drugs which can exhibit synergistic cytotoxic effect on cancer cells.
The liposome comprises of atleast 55% or more of phospholipid and the remaining
is cholesterol or a phospholpid derivatized with polyethyleneglycol. The
phospholipids claimed are one or a combination from the group consisting of
phosphatidylcholine, phosphatidylglycerol, phosphatidylserine and
sphingomyelin. Moreover, the formulation claimed is a single compartment vesicle. This invention involves both drugs being encapsulated in the same compartment leading to limited encapsulation efficiencies of the drugs. The vesicle does not show triggered release in response to multiple stimuli and deals with combinations of paclitaxel only with cisplatin or doxorubicin (one hydrophobic and one hydrophilic drug). The vesicle can be delivered only by intravenous and intraperitoneal routes.
US Patent 7,820,718 describes the combination chemotherapy of paclitaxel with ceramide (an apoptosis inducing sphingophospholipid). where paclitaxel is effected by cremophore-mediated delivery or liposome-mediated delivery, and the contacting with C.sub.6-ceramide is effected by cremophore-mediated delivery,

alcohol-mediated delivery or liposome-mediated delivery. This patent deals with a direct co-administration or simple single compartment conventional liposomes and involves encapsulation of the anticancer drug into the liposome while the ceramide is incorporated as a part of the liposomal bilayer. This invention does not deal with optimization of sustained release and the conventional liposomes used limit control over the release profiles and encapsulation efficiencies of the drugs.
US Patent 7,846,441 claims a method for the treatment of a human patient with a malignant progressing tumor or cancer characterized by overexpression of ErbB2 receptor, comprising administering a combination of an intact antibody which binds to epitope 4D5 within the ErbB2 extracellular domain sequence and a taxoid, in the absence of an anthracycline derivative, to the human patient in an amount effective to extend the time to disease progression. It involves combination of paclitaxel and anti-ErbB2 antibody against primary and metastatic breast cancer, where the agents are directly administered or delivered in an immunoliposome. This involves incorporation of paclitaxel in the liposomes while the antibody is attached at the surface of the immunoliposome. Immunoliposomes are known to have very short circulation times and do not show trigger specific release of the constituents.
US Patent 6,565,889, reports a bilayer structure encapsulating multiple containment units. It is a vesosome or multiple vesicle in vesicle system where the membrane encapsulates the multiple containment units, and is distinct from the multiple containment units. It does not deal with the codelivery of multiple drugs simultaneously nor does it include paclitaxel and cur cumin combinations. Further, it does not deal with multiple triggered release and is not nanosized.

OBJECT OF THE INVENTION
The main object of this invention is in providing a multicompartmental nano sized conjoined vesicle for delivery of multiple anticancer drugs which act synergistically in response to multiple biological stimuli like pH variation temperature and enzyme level
Another object of the invention is high encapsulation of multiple hydrophobic anticancer drugs for sustained release thereof into the body.
Yet another object of this invention is to provide drug delivery through administration by intranasal and aerosol routes apart from conventional intravenous method.
A further object of this invention is to provide nano vesicles of < 500 nm with increased accumulation of the drug in target specific areas when intravenously administered due to enhanced permeability and retention effect of the nanovesicles
BRIEF DESCRIPTION OF INVENTION
The invention consists of a conjoined lipid based, two or more compartment conjoined nanovesicles which can encapsulate two or more hydrophobic anticancer drugs and has a diameter less than 500 nm. This can result in an increased accumulation of the formulation specifically in the tumor region when given intravenously, due to enhanced permeability and retention (EPR) effect.

The invention consists of atleast two compartments or vesicles joined together. One of the compartments of the conjoined vesicle is composed of dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylethanolamine (DOPE) in a molar ratio of 6:4.while the other compartment of the conjoined vesicle is composed of palmitoyloleoylphosphatidylethanolamine (POPE), Cholesterol (CHOL) and a saturated temperature sensitive lipid which can be either distearoylphosphatidylcholine (DPPC or DSPC). DSPC/DPPC: POPE was maintained at the molar ratio of 9:1/8:2 and total phospholipid: CHOL molar ratio was maintained at 7:3. CHOL here acts as the glue that holds the two vesicles together by imparting strong hydrophobic forces at the junction. Due to the unique lipid composition of the nanovesicle system, it shows a temperature sensitivity, pH sensitivity and enzyme sensitivity in presence of secretory phospholipase A2 (sPLA2) enzyme which is prevalent in the tumor region. The system therefore exhibits triple triggered release of the drugs.
The invention has the following advantages or unique features
1. two distinct conjoined compartments within the vesicle for high encapsulation of two or more hydrophobic anticancer drugs
2. size less than 500nm diameter
3. triple triggered release in response to changes of pH, temperature and
phospholipase A2
4 passes blood brain barrier when given intranasally
5. maintains high airway patency when given as an aerosol
6. is actively endocytosed by the cancer cells
7. when loaded with two or more drugs, it has synergistic action of the two encapsulated hydrophobic drugs like paclitaxel and curcumin resulting in lower IC50 in various cancer cells in vitro

This invention relates to conjoined lipid based multi compartment nano vesicles
for encapsulating and delivering drugs comprises at least two separate vesicles
joined together wherein a first vesicle is composed of
dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylethanolamine
(DOPE) and a second vesicle composed of
palmitoyloleoylphosphatidylethanolamine (POPE), cholesterol (CHOL) and a temperature sensitive saturated lipid such as DPPC or distearoylphosphatidylcholine(DSPC)
This invention also relates to a process for preparing conjoined multicompartmental nanvesicle for delivery drugs which comprises the steps of (a) dissolving a lipid such as dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylethanolamine (DOPE) in organic solvents drying to obtain a thin lipid film, hydrating and centrifuging resuspending and sonicating the same to produce said first compartment (b) dissolving distearoylphosphatidylcholine (DSPC) or DPPC, palmitoyloleoylphosphatidylethanolamine (POPE), and cholesterol in an organic solvent drying the same to produce a thin film, hydrating said thin film the suspension of said first compartment obtained on sonification, centrifuging, resuspending obtained pellets and sonicating thereafter to obtain conjoined multicompartment nanovesicles.
DETAILED DESCRIPTION OF THE INVENTION
The invention is a conjoined lipid based, two or more compartment nanovesicle which can encapsulate two or more hydrophobic anticancer drugs for a synergistic effect in the treatment of cancers using combination chemotherapy.

The invention consists of a two compartment nanovesicle composed of dipalmitoylphosphatidylcholine, dioleoylphosphatidylethanolamine and paclitaxel in one compartment of the nanovesicle and dipalmitoylphosphatidylcholine. palmitoyloleoylphosphatidylethanolamine, cholesterol and curcumin in the second compartment of the same conjoined nanovesicle.
The invention was prepared as a two step process. The first compartment of the
nanovesicle is prepared by dissolving desired amounts of
dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylethanolamine
(DOPE) (6:4 molar ratio) in 2:1 chloroform: methanol mixture along with a
hydrophobic anticancer drug like paclitaxel. Paclitaxel was added to the lipid
mixture with paclitaxel: lipid molar ratio as 1:2 in the drug loaded versions of the
invention. The solution was then dried in a rotary evaporator under vacuum at
40°C to obtain a thin lipid film. The lipid film was hydrated with phosphate
buffered saline (PBS) (pH 7.4) solution in a rotary vacuum evaporator at about
200 rpm at 45°C for one hour. The suspension was then centrifuged at 25000 g for
15 min at 4°C and the pellet was resuspended in desired volume of PBS Following
this, the suspension was sonicated at 50% amplitude at 20 KHz for 2 minutes or
equivalent using a sonicator and kept as suspension for compartment 1. Desired
amounts of distearoylphosphatidylcholine (DSPC) /DPPC,
palmitoyloleoylphosphatidylethanolamine (POPE), cholesterol (CHOL) and curcumin were then weighed and dissolved in 2:1 chloroform: methanol to form the second compartment of the nanovesicle. Curcumin was used as the second hydrophobic drug in the drug loaded version of the invention. Curcumin to lipid molar ratio was maintained as 1:2. This solution of lipid and curcumin was dried in a rotary evaporator under vacuum at 40°C to obtain a thin lipid film of

compartment 2 of the nanovesicle. The lipid film was hydrated with the suspension for compartment 1 prepared as described by adding the desired volume of the suspension for compartment 1 to the thin film of compartment 2 prepared in a rotary vacuum evaporator and rotating the round bottom flask at about 200 rpm at 40°C for one hour. Molar ratio of the lipids constituting second compartment of the nanovesicle to lipids constituting first compartment was maintained as 2:1. The suspension thus obtained was again centrifuged at 25000 g for 15 min at 4°C and the pellet was resuspended in desired volume of PBS. Following this, the suspension was sonicated at 50% amplitude at 20 KHz for 1 minute or equivalent using a sonicator to prepare the conjoined nanovesicle. The above combinations of phospholipids are only illustrative and the dual compartment nanovesicle can be formed of any combinations of phospholipids and cholesterol along with at least two hydrophobic anticancer drugs. Further, targeting moieties like folic acid, transferrin, and image contrast agents like gold nanorods, iron oxide nanoparticles or fluorescent dyes can be co-encapsulated within the nanovesicle.
The invention with or without drugs has a particle size less than 500 nm as determined by dynamic light scattering (DLS) using laser particle analyzer (BI 200SM, Brookhaven Instruments Corporation). Polydispersity index suggested a uniform size distribution of the particles (Table 1). Transmission electron microscopy (TEM) as shown in figure 1 further confirmed the size and structure of the invention depicting two compartment conjoined nanovesicles. This interaction between the two is attributed to the presence of cholesterol (CHOL) in the second vesicle, which causes strong hydrophobic interactions at the interface. The nanovesicles were also characterized for surface charge by determining their zeta potential using zeta potential analyzer (ZetaPALS, Brookhaven Instruments Corporation). Zeta potential of the formulation was found to be highly negative (~

30 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.
The invention with or without drugs is surface active and maintains > 90% airway patency in a capillary surfactometer. Airway patency was studied using a capillary surfactometer and expressed in terms of % opening time of the capillary. As shown in figure 2, the invention in the absence and presence of drugs showed 99.7 ± 0.06 % capillary opening time as compared to the standard paclitaxel (Taxol®) and curcumin solution which exhibited 0.42 ±0.1 % and 0.32 ±0.19 % capillary opening time (p<0.05) for the entire observation period i.e. 120 seconds. This clearly indicates that the invention in the absence of any drugs is surface active, has good surfactant properties, and maintains the open state of the airways which are desirable for pulmonary delivery.
The invention in the absence of any drugs was actively endocytosed by the various cancer cells. Cellular uptake study for present invention was done on Hela (human cervical cancer) and A549 (human non small cell lung carcinoma) cell lines in order to study its uptake and internalization cancer cells. Calcein, dissolved in PBS was loaded into the first compartment during the first hydration and rhodamine-6G was loaded into the second compartment during second hydration. The invention was endocytosed by an ATP dependant mechanism. Mechanism of cellular uptake and interaction of invention with A549 cells was studied by incubating the cells with DV 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. Both the compartments were separately tagged for identification and studied for

their uptake mechanism. As shown in figure 3, in both the cases i.e. DV-1 and DV-2, 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.
The invention in the absence of any drugs passes the blood brain barrier on being administered intranasally. In order to assess the possibility of the invention to reach brain when administered intranasally, in vivo brain uptake study was done in female Wistar rats. Rhodamine-6G and calcein dyes were loaded in the two compartments of the formulation. It was observed that brain sections were showing fluorescence after 30 min., 2 h and 4h as shown in figures 4. The formulation was uniformly distributed in different parts of the brain through intranasal route and reached very rapidly to the brain-
The invention co-encapsulates multiple fluorescent dyes for tracking and imaging of the nanovesicle. The nanovesicles were imaged using confocal laser scanning microscopy (CLSM) with FITC-dextran in one of the compartments of the nanovesicle and cresol red loaded in the other compartment of the nanovesicle. The sample was then analyzed by confocal microscope. Both the dyes were visible in the same conjoined nanovesicle. This confirms that the conjoined nanovesicle can be tracked by fluorescent dyes.
Present invention when loaded with two hydrophobic drugs was found to have a high encapsulation efficiency of 80.7 ±5.1 % (n=5) for paclitaxel as determined by reverse-phase HPLC method (Agilent 1100 Binary LC pump liquid chromatograph, Zorbax SB C-18 column, 250x4.6mm, 5um). The mobile phase was acetonitrilewater (60: 40, v/v), injection volume was 20ul and the column

temperature maintained at 25°C. The analysis was performed at a flow rate of 1.5 ml/min with the UV detector at 227 nm. Curcumin too showed a high encapsulation of 76 ± 1% (n=3) as determined by fluorescence spectrophotometry using a Hitachi 250098. F2500 spectrophotometer at 420 nm excitation and 552 nm emission wavelengths. High encapsulation efficiency of drugs is quite crucial in designing an economic drug delivery system.
The invention when loaded with two hydrophobic drugs, shows a synergistic effect of the drugs in vitro eg paclitaxel and curcumin within the invention is more effective than standard drugs alone in many cancers when evaluated in vitro using the cytotoxicity and IC50 assay. In vitro cytotoxicity study of the invention when loaded with two hydrophobic drugs was done on HCT-15 (drug resistant human colon carcinoma), A549 (human non small cell lung carcinoma), B16F10 (murine melanoma), MDAMB-231 (drug resistant human breast carcinoma) and U87-MG (drug resistant human glioblastoma) cell lines in order to determine its antiproliferative efficacy. Inhibitory activity of DV encapsulating only paclitaxel (DV-PTX), DV encapsulating only curcumin (DV-CMN), DV encapsulating both the drugs (DV-PTX-CMN) and blank DV (DV-B) were compared with each other and with the inhibitory activity of Taxol® and curcumin. IC50 of the formulations as obtained as a result of their 72 hours exposure are given in Table 2. As can be seen, DV-PTX-CMN showed several folds less IC50 as compared to that of Taxol® and free curcumin (p<0.05). This indicates a clear advantage of our formulation over the currently marketed formulation of paclitaxel as well as free curcumin.
The invention is effective in bypassing the drug resistance in drug resistant cancers This is depicted by the low IC50 observed with the invention when loaded with

two hydrophobic drugs in Table 2, particularly in the cases of drug resistant cell lines like HCT-15, MDAMB-231 and U87-MG clearly suggest that present formulation bypasses P-glycoprotein pump, which is generally overexpressed in such drug resistant cell lines and efflux the drug out of the cell. The invention bypasses this efflux pump by acting as a shield for the drug such that the drug does not come into direct cellular recognition.
The invention exhibits a sustained drug release pattern of the encapsulated hydrophobic drugs for example paclitaxel as well as curcumin under physiological pH and temperature conditions. The formulation however showed a significantly higher (p<0.05) release of both the drugs at 42°C as compared to 37°C, at pH 5.5 as compared to pH 7.4 and in the presence of sPLA2 enzyme as compared to in its absence (figure 5). However, maximum release was obtained when all the three triggers i.e. low pH, high temperature and sPLA2 enzyme were used together (figure 5). This shows that the formulation is sensitive to three different trigger mechanisms and can therefore result in significantly higher release of the encapsulated drugs in the tumor region which often exhibits conditions such as low pH, high temperature and elevated extracellular sPLA2 levels.
The invention when loaded with two hydrophobic drugs, is effective as an aerosol in pulmonary metastasis of multiple melanoma and lung cancers. In-vivo efficacy study for the invention when loaded with two hydrophobic drugs as an aerosol, intended for the treatment of lung cancer was done on pulmonary metastasis model of murine melanoma (B16F10) developed in female C57B1/6 mice (6-8 weeks old). Percentage growth inhibition obtained in this efficacy study is given in figure 6(a). Taxol® showed an inhibition of 39% at 10 mg/kg dosage given once in three days. DV-PTX showed an inhibition of 52 %, DV-CMN showed an

inhibition of 47% and DV-PTX-CMN (the invention when loaded with paclitaxel and curcumin) showed the maximum growth inhibition of 80%. Figure 6(b) shows the lung weight of the animals in different groups as obtained after sacrifice. Lung weights of the animals treated with DV-PTX-CMN (invention when loaded with paclitaxel and curcumin) were found to be significantly less as compared to the other groups (p<0.05). Figures 7(a) and 7(b) show the total number of tumor nodules and percentage area of the lungs occupied by the tumor nodules respectively in different groups. It can be observed that the total number of nodules as well as the percentage lung area in the case of DV-PTX-CMN treated group (invention when loaded with paclitaxel and curcumin) was significantly less as compared to the other groups.
Table I below show physiochemical properties of the invented conjoined nanovesicles

Parameter Value
Hydrodynamic diameter (nm) 473.7 ±43.4
Polydispersity index 0.033 ±0.01
Zeta potential (mV) -29.4 ± 2.7
Table II shows IC50 (nM) and combination index for the invention when loaded with two hydrophobic drugs, on different cancer cell lines (n=3) A549 is the lung adenocarcinoma cell line, B16F10 is the multiple melanoma, HCT 15 is the drug resistant colon carcinoma, MDAMB231 is the drug resistant breast cancer cell line and U87 MG is the drug resistant gliobastoma. DV-PTX-CMN is the invention when loaded with two hydrophobic drugs, DV-PTX is the invention containing only one drug paclitaxel, DV-CMN is the invention containing only one drug curcumin, Taxol denotes the paclitaxel alone without any

nanovescile, curcumin denotes the free curcumin and DV-B indicates the two compartment nanovesicle without any drug.

Formula tion A549 B16F10 HCT-15 MDAMB-
231 U87-MG
DV-FIX-CMN 14.3=2.3 34.9 ± 12.3 25.2 ±5.3 10.1 ±2.4 43 ±4.5
DV-PTX 112.65 ±13.4 234.3 ± IS.5 342.43 ± 15.9 435.1 = 10.2 153±1S.5
DV-CMN 704.3 = 20.4 639.9 ±35.4 1137.63 ± 40.7 1027.3 ± 24.4 741 ±17.1
Taxol® 149.4 ±3.4 344.3 ±20.3 443.3 ± 28.4 673.2 ± 34.2 727 ±3.4
Curcumin S34.6 ± 23.5 987.4=10.3 1234.34 ±
49.3 1162.2 ± 53.5 S37±56.3
DV-B - - - - -
Combination Index 0.141 0.237 0.109 0.039 0.374
Description with reference to the drawings accompanying this text is given below. Figure 1: Transmission electron microscopy image of the invention without any drugs showing conjoined nanovesicles with two compartments joined together and having a size < 500 nm (Scale bar: 100 ran)
Figure 2: Percentage capillary opening time of the invention in the absence of any drugs shows 99% airway opening. (DV) which is maintained even on loading two hydrophobic drugs (DV-PTX-CMN). It has been compared with that of control drugs taxol and curcumin alone which are block the airways (n=3)
Figure 3: Active internalization of the invention without any drugs after incubation of A549 cells under normal and ATP depleted conditions. The invention was tagged in each of the compartments as shown in (a) and (b) for each compartment (n=3) The amount of the invention within the cells is depicted by rhodamin levels and is higher under ATP active (normal) conditions and lower under ATP depleted conditions.

Figure 4: Confocal laser microscopy image of brain tissue sections from frontal lobe after intranasal administration of the invention in mice after (i) 30 min (ii) 2 h (iii) 4h. indicating the ability of the invention without any drugs to cross the blood brain barrier
Figure 5(a) and (b) shows In vitro paclitaxel and curcumin release from the invention when loaded with two hydrophobic drugs. The top figure depicts the release of paclitaxel which is sustained over 48 hours and shows a triggered response in presence of pH 6, temperature of 42 C and presence of phospholipase A2 enzyme. The bottom figure depicts the release of curcumin which is sustained over 48 hours and shows a triggered response in presence of pH 6, temperature of 42 C and presence of phospholipase A2 enzyme.
Figure 6 (a) Percentage growth inhibition observed with the invention when loaded with two hydrophobic drugs and compared with suitable controls in the B16 flO multiple melanoma metastasis model in mice.
Figure 6 : (b) Lung weights as observed in multiple melanoma metastasis model after treatment with aerosols of the invention when loaded with two hydrophobic drugs and other control groups (n=6).
Figure 7: (a) and (b) Total number of nodules in the lungs observed after treatment with the invention when loaded with two hydrophobic drugs and compared with suitable controls in the B16F10 multiple melanoma meastasis model in mice (n=6). (b) % area of lungs occupied by tumor nodules as (n=6).
The following examples illustrate the invention without limiting the scope thereof

EXAMPLE 1
The invention was prepared as a two step process. The first compartment of the
nanovesicle is prepared by dissolving desired amounts of
dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylethanolamine
(DOPE) (6:4 molar ratio) in 2:1 chloroform: methanol mixture. The solution was
then dried in a rotary evaporator under vacuum at 40°C to obtain a thin lipid film.
The lipid film was hydrated with phosphate buffered saline (PBS) (pH 7.4)
solution in a rotary vacuum evaporator at about 200 rpm at 45°C for one hour. The
suspension was then centrifuged at 25000 g for 15 min at 4°C and the pellet was
resuspended in desired volume of PBS Following this, the suspension was
sonicated at 50% amplitude at 20 KHz for 2 minutes or equivalent using a
sonicator and kept as suspension for compartment 1. Desired amounts of
distearoylphosphatidylcholine (DSPC) /DPPC,
palmitoyloleoylphosphatidylethanolamine (POPE), cholesterol (CHOL) weighed and dissolved in 2:1 chloroform: methanol to form the second compartment of the nanovesicle. This solution of lipid was dried in a rotary evaporator under vacuum at 40°C to obtain a thin lipid film of compartment 2 of the nanovesicle. The lipid film was hydrated with the suspension for compartment 1 prepared as described by adding the desired volume of the suspension for compartment 1 to the thin film of compartment 2 prepared in a rotary vacuum evaporator and rotating the round bottom flask at about 200 rpm at 40°C for one hour. Molar ratio of the lipids constituting second compartment of the nanovesicle to lipids constituting first compartment was maintained as 2:1. The suspension thus obtained was again centrifuged at 25000 g for 15 min at 4°C and the pellet was resuspended in desired volume of PBS. Following this, the suspension was sonicated at 50% amplitude at 20 KHz for 1 minute or equivalent using a sonicator to prepare the conjoined nanovesicle.

EXAMPLE 2
The invention was prepared as described above but was loaded with two hydrophobic drugs. In the drug loaded version, one drug like paclitaxel and was added to the chloroform:methanol mixture containing lipids forming the first compartment. Paclitaxel was added to the lipid mixture with paclitaxel: lipid molar ratio as 1:2. Curcumin was used as the second hydrophobic drug and added to the lipid mixture forming the second compartment. Curcumin to lipid molar ratio was maintained as 1:2.
EXAMPLE 3
The invention in the absence or presence of drugs forms at least two compartment conjoined nanovesicles with a particle size less than 500 nm. Transmission electron microscopy (TEM) as shown in figure 1 confirms the size and structure of the invention depicting two compartment conjoined nanovesicles. TEM imaging was done by negative staining method using CM200 (Philips) transmission electron microscope operating at 120 kV.
EXAMPLE 4
The invention in the absence of drugs, when evaluated by dynamic light scattering, showed hydrodynamic diameters of 473 nm. The surface charge was determining as the zeta potential using zeta potential analyzer (ZetaPALS. Brookhaven Instruments Corporation). Zeta potential of the formulation was found to be highly negative (—30 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.

EXAMPLE 5
The invention in the absence of drugs was evaluated for its ability to maintain airway patency in a capillary surfactometer and expressed in terms of % opening time of the capillary. As shown in figure 2, DV alone without any drugs as well as when loaded with two hydrophobic drugs DV-PTX-CMN showed 99.7 ± 0.06 % capillary opening time, as compared to the standard paclitaxel formulation (Taxol®) and curcumin solution which exhibited 0.42 0.1 % and 0.32 ±0.19 % capillary opening time (p<0.05) for the entire observation period i.e. 120 seconds. This clearly indicates that the invention in the absence of any drugs is surface active, Yias good surfactant properties, and maintains the open state of trie airways which are desirable for pulmonary delivery.
EXAMPLE 6
The invention in the absence of drugs was endocytosed by an ATP dependant mechanism. Mechanism of cellular uptake and interaction of rhodamine-6G loaded formulation with A549 cells was studied by incubating the cells with DV 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 4°C temperature. Both the compartments were tagged for identification and studied for their uptaKe mechanism. As shown in figure 3. in both the cases i.e. DV-1 and DV-2 (different compartments tagged in each case), cells pretreated with 0.1 % sodium azide and cells incubated at 4°C showed significantly less (p<0.05) intracellular rhodamine-6G content at all time points as compared to those incubated under normal conditions, i.e. 37°C 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 4°C rather than 37°C. This suggests that the cellular uptake of the invention (DV) 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 4°C. Moreover, uptake of the invention was found to be a time dependent process as increase in intracellular rhodamine-6G content was observed with increasing time points.
EXAMPLE 7
The invention in the absence of drugs passes the blood brain barrier on being administered intranasaYry. In order to assess the possibility of the invention to reach brain when administered intranasally, in vivo brain uptake study was done in female Wistar rats. Rhodamine-6G and calcein dyes were used to track the invention. It was observed that brain sections were showing fluorescence after 30 min., 2 h and 4h as shown in figures 4. The invention in the absence of any drugs was uniformly distributed in different parts of the brain through intranasal route and reached very rapidly to the brain. Briefly, animals were anesthetized by giving ketamine (i.p) and were placed in the supine position in anesthesia chamber. 100 µl of the dye loaded sample was administered intranasally as 10 µl bolus given after every 2 minutes into alternating sides of the nasal cavity. Animals were sacrificed at time intervals of 30 min, 2 hr and 4 hr each and tissues from brain were analysed 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). From the study it can be inferred that the present

invention when given intranasally is capable of reaching brain without affecting other organs such as lungs.
EXAMPLE 8
The invention when loaded with two hydrophobic drugs has a high encapsulation efficiency >70% for both the drugs and shows a synergistic effect of the drugs for example paclitaxel and curcumin and is more effective than standard drugs alone in many cancers when evaluated in vitro using the cytotoxicity and IC50 assay. In vitro cytotoxicity study of the invention containing two drugs was done on HCT-15 (drug resistant human colon carcinoma), A549 (human non small cell lung carcinoma), B16F10 (murine melanoma), MDAMB-231 (drug resistant human breast carcinoma) and U87-MG (drug resistant human glioblastoma) cell lines in order to determine its antiproliferative efficacy. Inhibitory activity of DV encapsulating only paclitaxel (DV-PTX), DV encapsulating only curcumin (DV-CMN), DV encapsulating both the drugs (DV-PTX-CMN) and blank DV (DV-B) were compared with each other and with the inhibitory activity of Taxol® and curcumin. IC50 of the formulations as obtained as a result of their 72 hours exposure are given in table 2. As can be seen, DV-PTX-CMN showed several folds less IC50 as compared to that of Taxol® and free curcumin (/?<0.05). This indicates a clear advantage of our formulation over the currently marketed formulation of paclitaxel as well as free curcumin.
As one of the aims of the present invention is to offer the advantages of combination chemotherapy over single agent therapy, combination index (CI) for DV-PTX-CMN was determined using the classic isobologram equation of Chou and Talalay [3, 4]. The concentration for 50 % cell death (IC50) was used for the determination of CI. The combination indices for different cell lines are given in

table 2. The CI being less than one in all the cases confirmed a synergistic mechanism of action of the two agents i.e. paclitaxel and curcumin in DV-PTX-CMN. Due to several folds less IC50 of DV-PTX-CMN as compared to the other single agent therapy formulations and its combination index less than one, it can be inferred that the present invention offering the combination therapy of paclitaxel and curcumin coencapsulated in a multicompartment lipid nanovesicle offers a statistically significant edge over single agent chemotherapy.
The invention is effective in bypassing the drug resistance in drug resistant cancers This is depicted by the low IC50 observed with the invention is loaded with two hydrophobic drugs, and evaluated in drug resistant cancers as shown in Table 2, particularly in the cases of drug resistant cell lines like HCT-15, MDAMB-231 and U87-MG clearly suggest that present formulation bypasses P-glycoprotein pump, which is generally overexpressed in such drug resistant cell lines and efflux the drug out of the cell. The invention bypasses this efflux pump by acting as a shield for the drug such that the drug does not comes into direct cellular recognition.
EXAMPLE 9
The invention can co-encapsulate multiple fluorescent dyes for tracking and imaging of the nanovesicle! The nanovesicles were imaged using confocal laser scanning microscopy (CLSM) with FITC-dextran in one of the compartments of the nanovesicle and cresol red loaded in the other compartment of the nanovesicle. The sample was then analyzed by confocal microscope. The confocal images of the invention was viewed by cresol red filter and FITC-dextran filters and multiple fluorescence was observed in the same conjoined vesicles. This a;lllows the

encapsulation of two different dyes simultaneously and in a conjoined vesicle and allows the conjoined nanovesicle to be tracked by fluorescent dyes.
EXAMPLE 10
The invention when loaded with two hydrophobic drugs exhibits a sustained drug release pattern for both for example paclitaxel as well as curcumin under physiological pH and temperature conditions. The formulation however showed a significantly higher (p<0.05) release of both the drugs at 42oC as compared to 37oC, at pH 5.5 as compared to pH 7.4 and in the presence of sPLA2 enzyme as compared to in its absence (figure 5). However, maximum release was obtained when all the three triggers i.e. low pH, high temperature and sPLA2 enzyme were used together (figure 5). This shows that the formulation is sensitive to three different trigger mechanisms and can therefore result in significantly higher release of the encapsulated drugs in the tumor region which often exhibits conditions such as low pH. high temperature and elevated extracellular sPLA2 levels.
EXAMPLE 11
The invention when loaded with two hydrophobic drugs is effective as an aerosol in pulmonary metastasis of multiple melanoma and lung cancers. In-vivo efficacy study for the invention when loaded with two hydrophobic drugs as an aerosol, intended for the treatment of lung cancer was done on pulmonary metastasis model of murine melanoma (B16F10) developed in female C57B1/6 mice (6-8 weeks old). On day 1 after the cell inoculation, mice were randomly divided into five groups, each having six animals in it. One group was kept as control that received 0.9% saline (7 ml) by aerosol. Group A received DV-PTX-CMN at 0.5 mg PTX/ml (7ml) dosage given as aerosol, group B received DV-PTX at 0.5 mg

PTX/ml (7ml) dosage given as aerosol and group C received DV-CMN as aerosol, with a dosage of curcumin corresponding to that given in DV-PTX-CMN. All aerosol treatments were given 5 days a week, o.i.d for four weeks. Group D was given Taxol® intravenously (10 mg/kg) once in every three days for four weeks. Body weights were taken intermittently during the entire experiment. Animals were sacrificed on day when 50 % animals died in the saline control group. Lungs were resected, and weighed. Anticancer efficacy was evaluated on the basis of % growth inhibition of the tumor. Percentage growth inhibition obtained in this efficacy study is given in figure 6 (a). Taxol® showed an inhibition of 39% at 10 mg/kg dosage given once in three days. DV-PTX showed an inhibition of 52 %, DV-CMN showed an inhibition of 47% and DV-PTX-CMN (the invention when loaded with two drugs) showed the maximum growth inhibition of 80%. Figure 6 (b) shows the lung weight of the animals in different groups as obtained after sacrifice. Lung weights of the animals treated with DV-PTX-CMN (invention when loaded with two drugs) were found to be significantly less as compared to the other groups (p<0.05).
EXAMPLE 12
In-vivo efficacy study for the invention when loaded with two hydrophobic drugs and delivered as an aerosol, intended for the treatment of lung cancer was done on pulmonary metastasis model of murine melanoma (B16F10) developed in female C57B1/6 mice (6-8 weeks old). On day 1 after the cell inoculation, mice were randomly divided into five groups, each having six animals in it. One group was kept as control that received 0.9% saline (7 ml) by aerosol. Group A received DV-PTX-CMN at 0.5 mg PTX/ml (7ml) dosage given as aerosol, group B received DV-PTX at 0.5 mg PTX/ml (7ml) dosage given as aerosol and group C received DV-CMN as aerosol, with a dosage of curcumin corresponding to that given in

DV-PTX-CMN. All aerosol treatments were given 5 days a week, o.i.d for four weeks. Group D was given Taxol® intravenously (10 mg/kg) once in every three days for four weeks. Body weights were taken intermittently during the entire experiment. Animals were sacrificed on day when 50 % animals died in the saline control group. Lungs were resected, and weighed. Tumor nodules on the lungs were counted using stereomicroscope. Total number of nodules in each lung and percentage area of the lung occupied by the nodules was determined. Figures 7 (a) and 7 (b) show the total number of tumor nodules and percentage area of the lungs occupied by the tumor nodules respectively in different groups. It can be observed that the total number of nodules as well the percentage lung area in the case of DV-PTX-CMN treated group (invention when loaded with two drugs) was significantly less as compared to the other groups. Animals in saline control and Taxol® group showed severe toxicity and abnormality indications as well as mortality. The group to which the invention loaded with two drugs was administered did not show any visible signs of abnormality in the animals.
Obvious modifications and alterations known to persons skilled in the art are within the scope and monopoly of the appended claims.

We claim
1. Conjoined lipid based multi compartment nanovesicles for encapsulating and delivering drugs comprising at least two separate nano vesicles joined together wherein a first vesicle is composed of dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylethanolamine (DOPE) and a second vesicle composed of palmitoyloleoylphosphatidylethanolamine (POPE), cholesterol (CHOL) and a temperature sensitive saturated lipid such as DPPC or distearoylphosphatidylcholine (DSPC) and the first and second compartments are capable of containing pharmaceutically active compounds.
2. The conjoined lipid based multi compartment nanovesicles as claimed in claim 1, wherein the first and second compartments encapsulated with individual anti cancer drugs which act synergistically when delivered.
3. The conjoined lipid based multi compartment nanovesicles as claimed in claims 1 & 2 wherein the drug to lipid molar ratio is 1:2.
4. The conjoined lipid based multi compartment nanovesicles as claimed in claims 1 to 3 wherein DPPC : DOPE molar ratio is 6:4 and DSPC / DPPC :POPE and cholesterol with DSPC / DPPC : POPE is maintained at a ratio of 9:1/8:1.
5. The conjoined lipid based multi compartment nanovesicles as claimed in claims 1-4 wherein said nano vesicles have a diameter less than 500nm.

6. The conjoined lipid based multi compartment nanovesicles as claimed in claims 1-5 wherein said nano vesicles are surface active with > 90% airway patency
7. The conjoined lipid based multi compartment nanovesicles as claimed in claims 1 to 6 wherein the anti cancer drugs are paclitaxel and curcumin.
8. The conjoined lipid based multi compartment nanovesicles claimed in claims 1-7 wherehi tracking and imaging modulation such as fluorescent dyes are encapsulated within each nano vesicle.
9. A process for producing conjoined multicompartment nanvesicles for delivering drugs comprising the steps of dissolving a lipid such as dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylethanolamine (DOPE) in organic solvents drying to obtain a then lipid film hydrating and centrifuging resmpending and sonicating the same to produce said first compartment (b) dissolving distearoylphosphatidylcholine (DSPC) or DPPC, palmitoyloleoylphosphatidylethanolamine (POPE), and cholesterol in an organic solvent drying the same to produce a thin film, hydrating said thin film the suspension of said first compartment obtained on sonification, centrifuging, resuspending obtained pellets and sonicating thereafter to obtain conjoined multicompartment nanovesicles.
10. The process as claimed in claim 9 wherein two separate drugs are encapsulated during step (a) and step (b) to produce drug encapsulated nanovesicles.

11. The conjoined lipid based nanovesicles substantially as herein described and exemplified.
12. A process for producing conjoined nano vesicles substantially as herein described and exemplified.