Title of Invention	"A PROCESS FOR THE PREPARATION OF pH SENSITIVE, MUCOADHESIVE HYDROGEL NANOPARTICLES FOR SYSTEMIC DELIVERY OF WATER SOLUBLE DRUGS THROUGH ORAL ROUTE"
Abstract	A process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water soluble drugs through oral route comprising the steps of: step 1 -dissolving a surfactant in oil to obtain reverse micelles, step 2 -adding an aqueous solution of vinyl pyrrolidone (VP), n-isopropyl acrylamide (NIPAAM) and acrylic acid (AA) monomers in the molar ratio of 40:30:30 (VP:NIPAAM:AA), a cross-linking agent, initiator, activator and water to said reverse micelles to form an optically clear and transparent reverse micellar solution, step 3 -subjecting said solution to polymerization in the presence of an inert gas to obtain nanoparticles, having uniform size between 10 nm to 50 nm diameter with low polydispersity, step 4 -separating the nanoparticles from the surfactant by precipitating the surfactant with zinc sulphate, as zinc di-ethylhexylsulphosuccinate, step 5 -separating said nanoparticles from unreacted material by dialysis, step 6- lypholyzing the dialyzed solution containing the nano-particles to dry powder.

Title of Invention

"A PROCESS FOR THE PREPARATION OF pH SENSITIVE, MUCOADHESIVE HYDROGEL NANOPARTICLES FOR SYSTEMIC DELIVERY OF WATER SOLUBLE DRUGS THROUGH ORAL ROUTE"

Abstract

A process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water soluble drugs through oral route comprising the steps of: step 1 -dissolving a surfactant in oil to obtain reverse micelles, step 2 -adding an aqueous solution of vinyl pyrrolidone (VP), n-isopropyl acrylamide (NIPAAM) and acrylic acid (AA) monomers in the molar ratio of 40:30:30 (VP:NIPAAM:AA), a cross-linking agent, initiator, activator and water to said reverse micelles to form an optically clear and transparent reverse micellar solution, step 3 -subjecting said solution to polymerization in the presence of an inert gas to obtain nanoparticles, having uniform size between 10 nm to 50 nm diameter with low polydispersity, step 4 -separating the nanoparticles from the surfactant by precipitating the surfactant with zinc sulphate, as zinc di-ethylhexylsulphosuccinate, step 5 -separating said nanoparticles from unreacted material by dialysis, step 6- lypholyzing the dialyzed solution containing the nano-particles to dry powder.

Full Text	,' A PROCESS FOR THE PREPARATION OF pH SENSITIVE, MUCOADHESIVE HYDROGEL NANOPARTICLES FOR SYSTEMIC DELIVERY OF WATER / SOLUBLE DRUGS THROUGH ORAL ROUTE This invention relates to a process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water-soluble drugs through oral route. BACKGROUND Existing oral delivery vehicles such as liposomes, microspheres, microcapsules and other polymer based microparticulate systems are in use for a long time to deliver the drugs in systemic circulation. Oral delivery formulation are preferable over the other routes of administration in the sense that it is easy to use and does not require the help of any medical personnel. Although significant advances have been made in the controlled delivery of drugs through other routes, the application of controlled release technology through oral route is limited. Drug delivery through oral route is subjected to frequently changing environments in the gastrointestinal tract. During transit it passes from strongly acidic gastric part to weakly alkaline intestinal part of the digestive system. In addition GI tract has also been known to be capable of absorbing and translocating to the blood, mainly via intestinal lymphatics, a wide variety of particles ranging from inert matter such as polystyrene particles and carbon particles, to live bacteria and viruses. While the mechanism of uptake is not understood completely, it is generally accepted that follicle associated epithelium of Peyer's patches, particularly membranous antigen transporting cells (M-cells), are the major site of uptake. Three possible routes of entry of macromolecule and particles from undamaged intestine to the blood circulation have been identified as: (i) through the M cells, (ii) through the normal epithelial cells and by (iii) paracellular means. Gut associated lymphoid tissue (GALT) plays an important but not an exclusive role in the particle uptake mechanisms although these specialized region of the intestine are still the subject of intensive study to understand their origin, differences throughout the GI tract and their roles. The development of oral delivery formulation needs better understanding of the interactions between the carrier particles and GALT. It is from an understanding of uptake and translocation of naturally occurring particles so that we can design synthetic and semi-synthetic carrier particles. These carrier particles can be more selectively routed, absorbed and translocated from gut to blood so as to be an efficient oral delivery formulation of various water-soluble drugs, enzymes, vaccines and nucleotides^tc. From the various studies reported in the literature, it can be concluded that the factors responsible for paniculate uptake by intestinal mucosa are the particle size, their surface charges, surface hydrophobicity and the presence and absence of surface ligands. Absorption of polystyrene particles in the size range of 50nm to SOOOnm diameters by the intestinal mucosa showed that up to an extent of 30% of the dose are adhered and translocated by the smallest particles of 50nm size. Even 6-7% of these 50nm size particles accumulates in the liver, spleen, blood, bone marrow and kidney. These investigations indicate that the success of oral delivery formulations is dependent on the use of small size particles preferably particles of size below 50nm diameter. Similarly the surface feature of these particles also dictates absorption process. As for example, uptake of 50nm size polystyrene particles occurs through both GALT and to lesser extent through normal epithelial tissue. On the other hand, when tomato lectin molecules are covalently attached on the surface of these particles, the uptake is not achieved solely via lymphatic tissue but also to a large extent by normal enterocytes. This reinforces that jignificant uptake can be improved by using specific surface ligands such as tomato lectin and invasin molecule. Another interesting observation is that the uptake is considerably reduced when hydrophilic poloxamer molecules coat the particle surface, thereby emphasizing that the absorption is dictated by thej^urface hydrophobicity of the polystyrene particles. Several oral delivery formulations have been patented in which different types of strategies have been attempted for carrying the drug from gut to systemic circulation. Two US patents 5,620,708 and 5,702,727 (Amkraut et al) have disclosed a composition comprising an active agent carrier particle attached to a binding moiety, which binds specifically to a target molecule present on the surface of mammalian enterocytes. The binding moiety binds to the target molecule with a binding affinity sufficient to initiate endocytosis or phagocytosis of the paniculate active agent carrier, so that the carrier is absorbed by the enterocyte. The active agent is then released from the carrier to the host's systemic circulation. Limitation: The chemical conjugation between drug and the targeted molecule may lead to decrease in drug efficacy and in this way the transportation of considerable amount of drug to systemic circulation is not possible. The use of liposomal carrier for oral drug delivery has also been attempted (US Patent 5,762,904; Okada et al). Due to the inherent instability of these vesicles of organized lipids, these lipids have been polymerized to make the aggregate more stable. Limitation: Although an enhanced stability in the GI tract has been achieved by this method, no substantial improvement on the translocation efficiency of the vaccines from gut to the systemic circulation has been observed. A novel strategy has been taken by Simpson et al (US Patent 6,051,239) in which modified botulinum toxin has been used which is capable of translocating from gut to the general circulation. Limitation: The inventors have missed one important point and that is the residence time of the toxin has to be longer in the intestine for effective translocation. As a result, the efficacy of the method is not high. A patent (US patent 6,159,502; Russell-Jones et al) disclosed the use of microspheres. Microparticles are good media for entrapment of drugs and the polymeric materials used can be modulated according to the desired need of the drug and delivery strategy. Limitation: Microspheres, being large size entities, are not good carriers for drug targeting, particularly when the drug is required to be transported to the blood through oral route. From the above survey one can conclude that gastric uptake and translocation are neither exceptional nor unusual. The deciphering of the complex formula would definitely lead to achieving the therapeutic drug delivery through oral route. Among these complex processes it seems that particle size is of prime importance, which has to be regulated to the level down to 50nm and below. This is because it is not always necessary that translocation has to be made only through Peyer's patches. If the paniculate uptake can be induced by way of non-lymphoid intestinal tissues by regulating its size and surface hydrophobicity, then possibility of delivery through the oral route will no longer be conjectured as it is known to occur in the case of virus and bacteria. The object of this invention is to obviate the above limitations. Other objects of the instant invention are: to develop a process for preparation of stimuli responsive nanoparticles of hydrogel materials, either void or encapsulating water soluble compounds. to develop a process for preparation of nanoparticles of hydrogel polymers constituted of random copolymer that can entrap water soluble compounds to the maximum possible extent. to develop a process for preparation of nanoparticles of hydrogel copolymeric materials having entire crosslinked polymeric chains so that the release of encapsulated material can be controlled. to develop a process for preparation of nanoparticles of hydrogel copolymer loaded with water soluble compounds with hydrophobic chains extended at the outer surface the nanoparticles. to develop a process for preparation of nanoparticles of hydrogel polymers containing water soluble compounds in which one of the constitutes of the copolymer is acrylic acidjto render the nanoparticles mucoadhesive and pH sensitive so that these nanoparticles can be used safely even in drastic acidic medium in stomach and remain in the gut for longer period of time to develop a process for preparation of nanoparticles of hydrogel material loaded with water-soluble compounds and dispersed in aqueous solution, which are free from other unwanted or toxic material like unreacted monomers. to develop a process for the preparation of a smart hydrogel nanoparticles of copolymer material loaded with water soluble compound for maximum translocation from gut to blood during oral delivery treatment. To achieve the said objectives this invention provides process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water-soluble drugs through oral route comprising the steps of: step 1 -dissolving a surfactant in oil to obtain reverse micelles, step 2 -adding an aqueous solution of vinyl pyrrolidone (VP), n-isopropyl acrylamide (NIPAAM) and acrylic acid (AA) monomers in the molar ratio of 40:30:30 (VP:NIPAAM:AA), a cross-linking agent, initiator, activator and water to said reverse micelles to form an optically clear and transparent reverse micellar solution, step 3 -subjecting said solution to polymerization in the presence of an inert gas to obtain nanoparticles, having uniform size between 10 run to 50 run diameter with low polydispereity, step 4 -separating the nanoparticles from the surfactant by precipitating the surfactant with zinc sulphate, as zinc di-ethylhexylsulphosuccinate, step 5 -separating said nanoparticles from unreacted material by dialysis, step 6- lypholyzing the dialyzed solution containing the nano-particles to dry powder. The said monomers are vinyl pyrrolidone (VP), n-isopropyl acrylamide (NIPAAM) and acrylic acid (AA). The said monomers are in the molar ratio of 40:30:30 (VP:NIPAAM:AA), The said cross-linking agent is NN-methylene bis acrylamide (MBA). The monomeric cross-linking agent used is 0.3 - 1.2 % w/w of said monomers. The said initiator used is ammonium persulphate of 2 % w/w of monomers. The said activator used is ferrous ammonium sulphate of 0.07% w/w of monomers. The said surfactant used is sodium bis ethylhexylsulphosuccinate (Aerosol OT) and said oil is analkane. The said alkane is n-hexane. Water soluble compound to be entrapped into the nanoparticles is added in step 2 along with water depending upon the molecular size of said compound. 1-10% of the water soluble compound by weight of the polymeric material is encapsulated into said nanoparticles. The water-soluble compound is flourecein-isothiocyanate (FITC-dextran). The dialyzed nanoparticles entrapping water soluble compound is lyophilized to dry powder for further use. The nanoparticles have size anywhere between 10 nm to 50 nm diameter with low polydispersity. The said inert gas is nitrogen. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING The invention will now be described with reference to the accompanying figure Figure 1 shows the flow diagram for the preparation of nanoparticles loaded with drug. DETAILED DESCRIPTION OF THE INVENTION In accordance with the invention the aqueous core of the reverse micellar droplet is used as a nanoreactor for the preparation of nanoparticles. The sizes of the aqueous core of such droplet determine the size of the nanoparticles formed and therefore it is possible to prepare ultra small size nanoparticle of the hydrogel material in aqueous medium of the reverse micellar droplets. The aqueous phase is regulated in such a manner so as to keep the entire mixture in an optically transparent microemulsion phase. The range of the aqueous phase cannot be defined because this should depend on factors such as monomer, surfactant or polarity of oil and the only factor is that the system is in optically transparent microemulsion phase. in accordance with the present invention, the nanoparticles of smart hydrogel material having a size range upto 50nm, preferably a size anywhere between lOnm and 50nm diameter have been prepared. In accordance with this invention the aqueous core of reverse micellar droplet is effectively used as nanoreactor to prepare hydrogel nanoparticles encapsulating water-soluble drugs. The process of the present invention has achieved smart hydrogel nanoparticles of size down to lOnm diameter with near monodispersity. The surfactant, sodium bisethylhexylsulphosuccinate, or aerosol OT (AOT) is dissolved in n-hexane (usually 0.03M to 0.1M AOT-hexane), to form reverse micelles. Aqueous solutions of various monomers, vinyl pyrrolidone (VP), N-isopropylacrylamide (NIPAAM) and acrylic acid (AA), N N' methylene bis acrylamide as cross-linking agent, ammonium persulphate as initiator, ferrous ammonium sulphate as activator, and if necessary, water soluble compounds such as flourecein-isothiocyanate (FITC-dextran), are addeTTahd the"poiymerization is done in the presence of N2 gas. The said initiator used is 15 microliters of saturated solution of ammonium per shulphate (2% w/w of monomers) and the activator used is 20 micriliter is 0.05 % w/v of ferrous ammonium sulphate (0.07% w/w of monomers). The maximum amount of entrapped compound that can be dissolved in reverse micelles during polymerization process varies from compound to compound, and has to be determined by gradually increasing the amount till the clear reverse micelles is transformed to translucent solution. NI gas was passed through the reaction mixture and polymerization was done in N2 atmosphere. The nanoparticle formed in the reverse micelle remain dispersed in the organic solvent and are precipitated by adding ZnSCX solution, and the Zinc di-ethylhexylsulphosuccinate [Zn (DHESS)2] precipitate was pelleted by centrifugation at 8000 rpm. The Zn (DHESS)2 was dissolved in hexane and washed thrice with water. This water phase was collected and mixed to the supernatenant. The supematent was dialysed for 3 hours using 12 kD cut-off dialysis membrane. The dialyzed product was then lyophilized to dry powder for further use. Reference is now made to fig-1 of the accompanying drawing of the nanoparticles using reverse micelles. Reverse micellar droplets were prepared by dissolving the surfactant in oil. In step A, monomers, cross-linking agent, water-soluble compound such as drug, initiator and activator were added to the reverse micellar droplets. Polymerization was carried out in Na atmosphere to form copolymer nanoparticles encapsulating the water-soluble compound as shown in step B. In step C; the oil was removed by evaporation to get a dry mass, which contain drug encapsulated nanoparticles. This dry mass was resuspended in water and to it 30% ZnSO4 solution was added to precipitate out the surfactant as Zinc ethylhexylsulphosuccinateas Zn (DEHSS)2 as shown in the step D. In Step E centrifugation at 8000 rpm for 10 minutes was done to remove the precipitated surfactant and the supernatant liquid containing the nanoparticles was decanted off. The precipitate of Zn (DEHSS)2 was dissolved in oil and then leached with 1ml of water three times, and this leached solution was added to the supernatant liquid containing the nanoparticles. In step F the whole solution was dialyzed for three hours to remove the unreacted materials. Finally in step G the dialyzed solution was freeze-dried to get the nanoparticles in powder form. Normally 0.01-0.1 M AOT in n-hexane is used. Mixture of VP, NIPAAM and AA are used as monomers as they form water-soluble hydrogel materials on polymerization which are pH and thermosensitive. The cross-linking is done with MBA. Loading of the drug or any water-soluble molecule should be between 1% to 10% w/w of the polymer according to the solubility of the drug in the micellar system but it can also be increased if the solubility of the drug in the reverse micelles is high. EXAMPLES: The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention. Example-1 Preparation of placebo nanoparticles- In 40 ml of 0.03 M AOT solution in hexanM.3.2 of freshly distilled VP, 30 M,! of freshly distilled Acrylic acid and 208 \|ul of NIPAAM solution in water (228 mg/ml), 50^1 of MBA (0.49 mg/ml), 10 jil of 1% FAS solution and 100 \i 1 of water were added The solution was homogenous and optically transparent. The solution was then purged with NI gas for half an hour and 15^,1 of saturated solution of APS was added. Polymerization was done in presence of N2 gas at 35°C for 8 hour in a water bath with continuous stirring. The nanoparticles of random copolymer of VP + NIPAAM + AA was formed in the aqueous core of the reverse micellar system. Hexane was evaporated off in a rotary evaporator, and the dry mass resuspended in 15ml of distilled water. 2 ml of 30% ZnSO4 solution was added to the suspension, and the Zinc di-ethylhexylsulphosuccinate [Zn (DHESS^] precipitate was pelleted by centrifugation at 8000 rpm. The Zn (DHESS)2 was dissolved in hexane and washed thrice with water. This water phase was collected and mixed to the supernatenant. The supernatent was dialysed for 3 hours using 12 kD cut-off dialysis membrane. The dialyzed product was then lyophilized to dry powder for further use. Entrapment efficiency: (i) The entrapment efficiency of the FITC- dextran in copolymeric micelle was done as follows: The aqueous acidic extract after separation of nanoparticles including the repeated washing was collected and was made upto the volume of 10 ml. To a 100 M,! of that solution, 500 JA! of PBS( pH-8.0) was added and the concentration of the FITC-dextran was measured at 493 nm. The amount of FITC-dextran was calculated from the standard curve. The total amount of FITC-dextran left in the aqueous extract was substracted from the given amount of FITC-dextran originally added in the reaction and the entrapment efficiency was measured from the ratio of the amount of FITC-dextran entrapped to the total amount of FITC-dextran added x 100. The entrapment efficiency was found to be approximately 90%. , We claim: 1. A process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water soluble drugs through oral route comprising the steps of: step 1 -dissolving a surfactant in oil to obtain reverse micelles, step 2 -adding an aqueous solution of vinyl pyrrolidone (VP), n-isopropyl acrylamide (NIPAAM) and acrylic acid (AA) monomers in the molar ratio of 40:30:30 (VP:NIPAAM:AA), a cross-linking agent, initiator, activator and water to said reverse micelles to form an optically clear and transparent reverse micellar solution, step 3 -subjecting said solution to polymerization in the presence of an inert gas to obtain nanoparticles, having uniform size between 10 nm to 50 nm diameter with low polydispersity, step 4 -separating the nanoparticles from the surfactant by precipitating the surfactant with zinc sulphate, as zinc di-ethylhexylsulphosuccinate, step 5 -separating said nanoparticles from unreacted material by dialysis, step 6- lypholyzing the dialyzed solution containing the nano-particles to dry powder. 2. A process as claimed in claim 1 wherein said cross-linking agent is N, N-methylene bis acrylamide (MBA). 3. A process as claimed in the preceding claims wherein the monomeric cross-linking agent used is 0.3 - 1.2 % w/w of said monomers. 4. A process as claimed in claim 1 wherein said initiator used is ammonium per sulphate of 2 % w/w of monomers. 5. A process as claimed in claim 1 wherein said activator used is ferrous ammonium sulphate of 0.07% w/w of monomers. 6. A process as claimed in claim 1 wherein said surfactant used is sodium bis ethylhexylsulphosuccinate (Aerosol OT) and said oil is an alkane. 7. A process as claimed in claim 8 wherein 0.03 M to 0.1 M sodium bis ethylhexylsulphosuccinate in an alkane is used as a reverse micelles. 8. A process as claimed in claims 8 & 9 wherein said alkane is n-hexane. 9. A process as claimed in claim 1 wherein a water-soluble compound to be entrapped into the nanoparticles is added in step 2 along with water depending upon the molecular size of said compound. 10. A process as claimed in claims 1 & 11 wherein 1-10% of the water soluble compound by weight of the polymeric material is encapsulated into said nanoparticles. 11. A process as claimed in claim 1 wherein the water-soluble compound is flourecein- isothiocyanate (FTTC-dextran). 12. A process as claimed in claim 11 & 12 wherein the dialyzed nanoparticles entrapping water soluble compound is lyophilized to dry powder for further use. 13. A process as claimed in claim 1 wherein said inert gas is nitrogen. 14. A process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water-soluble drugs through oral route substantially as herein described with reference to the foregoing examples.

Full Text

,'
A PROCESS FOR THE PREPARATION OF pH SENSITIVE, MUCOADHESIVE HYDROGEL NANOPARTICLES FOR SYSTEMIC DELIVERY OF WATER
/
SOLUBLE DRUGS THROUGH ORAL ROUTE
This invention relates to a process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water-soluble drugs through oral route.
BACKGROUND
Existing oral delivery vehicles such as liposomes, microspheres, microcapsules and other polymer based microparticulate systems are in use for a long time to deliver the drugs in systemic circulation. Oral delivery formulation are preferable over the other routes of administration in the sense that it is easy to use and does not require the help of any medical personnel. Although significant advances have been made in the controlled delivery of drugs through other routes, the application of controlled release technology through oral route is limited.
Drug delivery through oral route is subjected to frequently changing environments in the gastrointestinal tract. During transit it passes from strongly acidic gastric part to weakly alkaline intestinal part of the digestive system. In addition GI tract has also been known to be capable of absorbing and translocating to the blood, mainly via intestinal lymphatics, a wide variety of particles ranging from inert matter such as polystyrene particles and carbon particles, to live bacteria and viruses. While the mechanism of uptake is not understood completely, it is generally accepted that follicle associated epithelium of Peyer's patches, particularly membranous antigen transporting cells (M-cells), are the major site of uptake. Three possible routes of entry of macromolecule and particles from undamaged intestine to the blood circulation have been identified as:
(i) through the M cells, (ii) through the normal epithelial cells and by (iii) paracellular means.
Gut associated lymphoid tissue (GALT) plays an important but not an exclusive role in the particle uptake mechanisms although these specialized region of the intestine are still the subject of intensive study to understand their origin, differences throughout the GI tract and their roles. The development of oral delivery formulation needs better understanding of the

interactions between the carrier particles and GALT. It is from an understanding of uptake and translocation of naturally occurring particles so that we can design synthetic and semi-synthetic carrier particles. These carrier particles can be more selectively routed, absorbed and translocated from gut to blood so as to be an efficient oral delivery formulation of various water-soluble drugs, enzymes, vaccines and nucleotides^tc.
From the various studies reported in the literature, it can be concluded that the factors responsible for paniculate uptake by intestinal mucosa are the particle size, their surface charges, surface hydrophobicity and the presence and absence of surface ligands. Absorption of polystyrene particles in the size range of 50nm to SOOOnm diameters by the intestinal mucosa showed that up to an extent of 30% of the dose are adhered and translocated by the smallest particles of 50nm size. Even 6-7% of these 50nm size particles accumulates in the liver, spleen, blood, bone marrow and kidney. These investigations indicate that the success of oral delivery formulations is dependent on the use of small size particles preferably particles of size below 50nm diameter.
Similarly the surface feature of these particles also dictates absorption process. As for example, uptake of 50nm size polystyrene particles occurs through both GALT and to lesser extent through normal epithelial tissue. On the other hand, when tomato lectin molecules are covalently attached on the surface of these particles, the uptake is not achieved solely via lymphatic tissue but also to a large extent by normal enterocytes. This reinforces that jignificant uptake can be improved by using specific surface ligands such as tomato lectin and invasin molecule. Another interesting observation is that the uptake is considerably reduced when hydrophilic poloxamer molecules coat the particle surface, thereby emphasizing that the absorption is dictated by thej^urface hydrophobicity of the polystyrene particles.
Several oral delivery formulations have been patented in which different types of strategies have been attempted for carrying the drug from gut to systemic circulation. Two US patents 5,620,708 and 5,702,727 (Amkraut et al) have disclosed a composition comprising an active agent carrier particle attached to a binding moiety, which binds specifically to a target molecule present on the surface of mammalian enterocytes. The binding moiety binds to the target molecule with a binding affinity sufficient to initiate endocytosis or phagocytosis of the

paniculate active agent carrier, so that the carrier is absorbed by the enterocyte. The active agent is then released from the carrier to the host's systemic circulation.
Limitation: The chemical conjugation between drug and the targeted molecule may lead to decrease in drug efficacy and in this way the transportation of considerable amount of drug to systemic circulation is not possible.
The use of liposomal carrier for oral drug delivery has also been attempted (US Patent 5,762,904; Okada et al). Due to the inherent instability of these vesicles of organized lipids, these lipids have been polymerized to make the aggregate more stable.
Limitation: Although an enhanced stability in the GI tract has been achieved by this method, no substantial improvement on the translocation efficiency of the vaccines from gut to the systemic circulation has been observed.
A novel strategy has been taken by Simpson et al (US Patent 6,051,239) in which modified botulinum toxin has been used which is capable of translocating from gut to the general circulation.
Limitation: The inventors have missed one important point and that is the residence time of the toxin has to be longer in the intestine for effective translocation. As a result, the efficacy of the method is not high.
A patent (US patent 6,159,502; Russell-Jones et al) disclosed the use of microspheres. Microparticles are good media for entrapment of drugs and the polymeric materials used can be modulated according to the desired need of the drug and delivery strategy.
Limitation: Microspheres, being large size entities, are not good carriers for drug targeting, particularly when the drug is required to be transported to the blood through oral route.
From the above survey one can conclude that gastric uptake and translocation are neither exceptional nor unusual. The deciphering of the complex formula would definitely lead to achieving the therapeutic drug delivery through oral route.

Among these complex processes it seems that particle size is of prime importance, which has to be regulated to the level down to 50nm and below. This is because it is not always necessary that translocation has to be made only through Peyer's patches. If the paniculate uptake can be induced by way of non-lymphoid intestinal tissues by regulating its size and surface hydrophobicity, then possibility of delivery through the oral route will no longer be conjectured as it is known to occur in the case of virus and bacteria.
The object of this invention is to obviate the above limitations.
Other objects of the instant invention are:
to develop a process for preparation of stimuli responsive nanoparticles of
hydrogel materials, either void or encapsulating water soluble compounds.
to develop a process for preparation of nanoparticles of hydrogel polymers
constituted of random copolymer that can entrap water soluble compounds to
the maximum possible extent.
to develop a process for preparation of nanoparticles of hydrogel copolymeric
materials having entire crosslinked polymeric chains so that the release of
encapsulated material can be controlled.
to develop a process for preparation of nanoparticles of hydrogel copolymer
loaded with water soluble compounds with hydrophobic chains extended at the
outer surface the nanoparticles.
to develop a process for preparation of nanoparticles of hydrogel polymers
containing water soluble compounds in which one of the constitutes of the
copolymer is acrylic acidjto render the nanoparticles mucoadhesive and pH
sensitive so that these nanoparticles can be used safely even in drastic acidic
medium in stomach and remain in the gut for longer period of time
to develop a process for preparation of nanoparticles of hydrogel material
loaded with water-soluble compounds and dispersed in aqueous solution,
which are free from other unwanted or toxic material like unreacted
monomers.
to develop a process for the preparation of a smart hydrogel nanoparticles of
copolymer material loaded with water soluble compound for maximum
translocation from gut to blood during oral delivery treatment.

To achieve the said objectives this invention provides process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles for systemic delivery of water-soluble drugs through oral route comprising the steps of:
step 1 -dissolving a surfactant in oil to obtain reverse micelles,
step 2 -adding an aqueous solution of vinyl pyrrolidone (VP), n-isopropyl acrylamide
(NIPAAM) and acrylic acid (AA) monomers in the molar ratio of 40:30:30
(VP:NIPAAM:AA), a cross-linking agent, initiator, activator and water to said
reverse micelles to form an optically clear and transparent reverse micellar
solution, step 3 -subjecting said solution to polymerization in the presence of an inert gas to
obtain nanoparticles, having uniform size between 10 run to 50 run diameter
with low polydispereity, step 4 -separating the nanoparticles from the surfactant by precipitating the surfactant
with zinc sulphate, as zinc di-ethylhexylsulphosuccinate, step 5 -separating said nanoparticles from unreacted material by dialysis, step 6- lypholyzing the dialyzed solution containing the nano-particles to dry powder.
The said monomers are vinyl pyrrolidone (VP), n-isopropyl acrylamide (NIPAAM) and
acrylic acid (AA).
The said monomers are in the molar ratio of 40:30:30 (VP:NIPAAM:AA),
The said cross-linking agent is NN-methylene bis acrylamide (MBA).
The monomeric cross-linking agent used is 0.3 - 1.2 % w/w of said monomers.
The said initiator used is ammonium persulphate of 2 % w/w of monomers.
The said activator used is ferrous ammonium sulphate of 0.07% w/w of monomers.
The said surfactant used is sodium bis ethylhexylsulphosuccinate (Aerosol OT) and said oil is analkane.

The said alkane is n-hexane.
Water soluble compound to be entrapped into the nanoparticles is added in step 2 along with water depending upon the molecular size of said compound.
1-10% of the water soluble compound by weight of the polymeric material is encapsulated into said nanoparticles.
The water-soluble compound is flourecein-isothiocyanate (FITC-dextran).
The dialyzed nanoparticles entrapping water soluble compound is lyophilized to dry powder for further use.
The nanoparticles have size anywhere between 10 nm to 50 nm diameter with low
polydispersity.
The said inert gas is nitrogen.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The invention will now be described with reference to the accompanying figure
Figure 1 shows the flow diagram for the preparation of nanoparticles loaded with drug.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention the aqueous core of the reverse micellar droplet is used as a nanoreactor for the preparation of nanoparticles. The sizes of the aqueous core of such droplet determine the size of the nanoparticles formed and therefore it is possible to prepare ultra small size nanoparticle of the hydrogel material in aqueous medium of the reverse micellar droplets. The aqueous phase is regulated in such a manner so as to keep the entire mixture in an optically transparent microemulsion phase. The range of the aqueous phase
cannot be defined because this should depend on factors such as monomer, surfactant or polarity of oil and the only factor is that the system is in optically transparent microemulsion phase.

in accordance with the present invention, the nanoparticles of smart hydrogel material having a size range upto 50nm, preferably a size anywhere between lOnm and 50nm diameter have been prepared.
In accordance with this invention the aqueous core of reverse micellar droplet is effectively used as nanoreactor to prepare hydrogel nanoparticles encapsulating water-soluble drugs. The process of the present invention has achieved smart hydrogel nanoparticles of size down to lOnm diameter with near monodispersity.
The surfactant, sodium bisethylhexylsulphosuccinate, or aerosol OT (AOT) is dissolved in n-hexane (usually 0.03M to 0.1M AOT-hexane), to form reverse micelles. Aqueous solutions of various monomers, vinyl pyrrolidone (VP), N-isopropylacrylamide (NIPAAM) and acrylic acid (AA), N N' methylene bis acrylamide as cross-linking agent, ammonium persulphate as initiator, ferrous ammonium sulphate as activator, and if necessary, water soluble compounds such as flourecein-isothiocyanate (FITC-dextran), are addeTTahd the"poiymerization is done in the presence of N2 gas. The said initiator used is 15 microliters of saturated solution of ammonium per shulphate (2% w/w of monomers) and the activator used is 20 micriliter is 0.05 % w/v of ferrous ammonium sulphate (0.07% w/w of monomers).
The maximum amount of entrapped compound that can be dissolved in reverse micelles during polymerization process varies from compound to compound, and has to be determined by gradually increasing the amount till the clear reverse micelles is transformed to translucent solution. NI gas was passed through the reaction mixture and polymerization was done in N2 atmosphere. The nanoparticle formed in the reverse micelle remain dispersed in the organic solvent and are precipitated by adding ZnSCX solution, and the Zinc di-ethylhexylsulphosuccinate [Zn (DHESS)2] precipitate was pelleted by centrifugation at 8000 rpm. The Zn (DHESS)2 was dissolved in hexane and washed thrice with water. This water phase was collected and mixed to the supernatenant. The supematent was dialysed for 3 hours using 12 kD cut-off dialysis membrane. The dialyzed product was then lyophilized to dry powder for further use.
Reference is now made to fig-1 of the accompanying drawing of the nanoparticles using reverse micelles. Reverse micellar droplets were prepared by dissolving the surfactant in oil. In step A, monomers, cross-linking agent, water-soluble compound such as drug, initiator and

activator were added to the reverse micellar droplets. Polymerization was carried out in Na atmosphere to form copolymer nanoparticles encapsulating the water-soluble compound as shown in step B. In step C; the oil was removed by evaporation to get a dry mass, which contain drug encapsulated nanoparticles. This dry mass was resuspended in water and to it 30% ZnSO4 solution was added to precipitate out the surfactant as Zinc ethylhexylsulphosuccinateas Zn (DEHSS)2 as shown in the step D. In Step E centrifugation at 8000 rpm for 10 minutes was done to remove the precipitated surfactant and the supernatant liquid containing the nanoparticles was decanted off. The precipitate of Zn (DEHSS)2 was dissolved in oil and then leached with 1ml of water three times, and this leached solution was added to the supernatant liquid containing the nanoparticles. In step F the whole solution was dialyzed for three hours to remove the unreacted materials. Finally in step G the dialyzed solution was freeze-dried to get the nanoparticles in powder form.
Normally 0.01-0.1 M AOT in n-hexane is used. Mixture of VP, NIPAAM and AA are used as monomers as they form water-soluble hydrogel materials on polymerization which are pH and thermosensitive. The cross-linking is done with MBA. Loading of the drug or any water-soluble molecule should be between 1% to 10% w/w of the polymer according to the solubility of the drug in the micellar system but it can also be increased if the solubility of the drug in the reverse micelles is high.
EXAMPLES:
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention.
Example-1
Preparation of placebo nanoparticles-
In 40 ml of 0.03 M AOT solution in hexanM.3.2 of freshly distilled VP, 30 M,! of freshly distilled Acrylic acid and 208 |ul of NIPAAM solution in water (228 mg/ml), 50^1 of MBA (0.49 mg/ml), 10 jil of 1% FAS solution and 100 \i 1 of water were added The solution was homogenous and optically transparent. The solution was then purged with NI gas for half an hour and 15^,1 of saturated solution of APS was added. Polymerization was done in presence of N2 gas at 35°C for 8 hour in a water bath with continuous stirring. The nanoparticles of

random copolymer of VP + NIPAAM + AA was formed in the aqueous core of the reverse micellar system. Hexane was evaporated off in a rotary evaporator, and the dry mass resuspended in 15ml of distilled water. 2 ml of 30% ZnSO4 solution was added to the suspension, and the Zinc di-ethylhexylsulphosuccinate [Zn (DHESS^] precipitate was pelleted by centrifugation at 8000 rpm. The Zn (DHESS)2 was dissolved in hexane and washed thrice with water. This water phase was collected and mixed to the supernatenant. The supernatent was dialysed for 3 hours using 12 kD cut-off dialysis membrane. The dialyzed product was then lyophilized to dry powder for further use.
Entrapment efficiency:
(i) The entrapment efficiency of the FITC- dextran in copolymeric micelle was done as follows: The aqueous acidic extract after separation of nanoparticles including the repeated washing was collected and was made upto the volume of 10 ml. To a 100 M,! of that solution, 500 JA! of PBS( pH-8.0) was added and the concentration of the FITC-dextran was measured at 493 nm. The amount of FITC-dextran was calculated from the standard curve. The total amount of FITC-dextran left in the aqueous extract was substracted from the given amount of FITC-dextran originally added in the reaction and the entrapment efficiency was measured from the ratio of the amount of FITC-dextran entrapped to the total amount of FITC-dextran added x 100. The entrapment efficiency was found to be approximately 90%. ,

We claim:
1. A process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles
for systemic delivery of water soluble drugs through oral route comprising the steps
of:
step 1 -dissolving a surfactant in oil to obtain reverse micelles,
step 2 -adding an aqueous solution of vinyl pyrrolidone (VP), n-isopropyl acrylamide
(NIPAAM) and acrylic acid (AA) monomers in the molar ratio of 40:30:30
(VP:NIPAAM:AA), a cross-linking agent, initiator, activator and water to said
reverse micelles to form an optically clear and transparent reverse micellar
solution, step 3 -subjecting said solution to polymerization in the presence of an inert gas to
obtain nanoparticles, having uniform size between 10 nm to 50 nm diameter
with low polydispersity, step 4 -separating the nanoparticles from the surfactant by precipitating the surfactant
with zinc sulphate, as zinc di-ethylhexylsulphosuccinate, step 5 -separating said nanoparticles from unreacted material by dialysis, step 6- lypholyzing the dialyzed solution containing the nano-particles to dry powder.
2. A process as claimed in claim 1 wherein said cross-linking agent is N, N-methylene
bis acrylamide (MBA).
3. A process as claimed in the preceding claims wherein the monomeric cross-linking
agent used is 0.3 - 1.2 % w/w of said monomers.

4. A process as claimed in claim 1 wherein said initiator used is ammonium per sulphate
of 2 % w/w of monomers.
5. A process as claimed in claim 1 wherein said activator used is ferrous ammonium
sulphate of 0.07% w/w of monomers.
6. A process as claimed in claim 1 wherein said surfactant used is sodium bis
ethylhexylsulphosuccinate (Aerosol OT) and said oil is an alkane.

7. A process as claimed in claim 8 wherein 0.03 M to 0.1 M sodium bis
ethylhexylsulphosuccinate in an alkane is used as a reverse micelles.
8. A process as claimed in claims 8 & 9 wherein said alkane is n-hexane.
9. A process as claimed in claim 1 wherein a water-soluble compound to be entrapped
into the nanoparticles is added in step 2 along with water depending upon the
molecular size of said compound.
10. A process as claimed in claims 1 & 11 wherein 1-10% of the water soluble compound
by weight of the polymeric material is encapsulated into said nanoparticles.
11. A process as claimed in claim 1 wherein the water-soluble compound is flourecein-
isothiocyanate (FTTC-dextran).

12. A process as claimed in claim 11 & 12 wherein the dialyzed nanoparticles entrapping
water soluble compound is lyophilized to dry powder for further use.
13. A process as claimed in claim 1 wherein said inert gas is nitrogen.
14. A process for the preparation of pH sensitive, mucoadhesive hydrogel nanoparticles
for systemic delivery of water-soluble drugs through oral route substantially as herein
described with reference to the foregoing examples.

Documents:

613-del-2003-abstract.pdf

613-del-2003-claims.pdf

613-del-2003-complete specification (granted).pdf

613-del-2003-correspondence-others.pdf

613-del-2003-correspondence-po.pdf

613-del-2003-description (complete).pdf

613-del-2003-drawings.pdf

613-del-2003-form-1.pdf

613-del-2003-form-19.pdf

613-del-2003-form-2.pdf

613-del-2003-form-3.pdf

613-del-2003-pa.pdf

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Patent Number

217609

Indian Patent Application Number

613/DEL/2003

PG Journal Number

29/2008

Publication Date

26-Sep-2008

Grant Date

27-Mar-2008

Date of Filing

16-Apr-2003

Name of Patentee

UNIVERSITY OF DELHI,

Applicant Address

DEPARTMENT OF CHEMISTRY, DELHI-110007, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	PROF. MAITRA, AMARNATH	UNIVERSITY OF DELHI, DEPARTMENT OF CHEMISTRY, DELHI-110007, INDIA.
2	MOZUMDAR, SUBHO	UNIVERSITY OF DELHI, DEPARTMENT OF CHEMISTRY, DELHI-110007, INDIA.
3	MITRA, SUSMITA	UNIVERSITY OF DELHI, DEPARTMENT OF CHEMISTRY, DELHI-110007, INDIA.
4	BHARALI, DHRUBA JYOTI	UNIVERSITY OF DELHI, DEPARTMENT OF CHEMISTRY, DELHI-110007, 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