Title of Invention

"A PROCESS FOR THE PREPARATION OF HIGHLY MONODISPERSED POLYMERIC HYDROPHILIC NANOPARTICLES"

Abstract

This invention relates to a process for the preparation of highly monodispersed polymeric hydrophillic nanoparticles without targeted materials having size of upto l00nm comprising in the steps of:- (i) dissolving a surfactant such as sodium bis ethyl hexyl sulphosuccinate in oil to obtain reverse micelles; (ii) adding an aqueous solution of a monomer or preformed polymer such as herein described to said reverse micelles and a crosslinking agent, initator and drug or a target substance, if required; (iii) subjecting such a mixture to the step of polymerization; (iv) drying the polymerized reaction product for removal of solvent to obtain dry nanoparticles and surfactant; (v) dispersing the dry mass in aqueous buffer; and (vi) separating the surfactant and other toxic materials therefrom.

Full Text

FIELD OIL INVENTION. This invention relates to a process for the preparation of highly monodispersed polymeric hy d r o p h i 1 i c na no pa r t i c 1 es without target molecules encapsulated therein and having sizes of upto 100 nm and a high monodispersity. The target materials are t hose o t he r t ha n t her a peu t i c o r b i o dine materials. OBJECTS,, OF. THE INVENTION A fi o b. j e c t o f t h is invention is to p r o p o s e a novel process for the preparation of highly monod i s pe r sed po1yme r i c na no pa r t i c1es with or wit hou t targeted materials other than therapeutic or bioactive materials and having a utiliize of upto 100 nm with a high monodispersi ty. A further object of this invention is to propose a process for the preparation of said polymeric monopartides capable of being modulated to r e qu i r e d s i zes . Another ob.ject of this invention is to propose a process for the preparation of said highly monodispersed polymeric nanoparticles of subcollodial size with or without targeted materials other than t h e r a p e u t i c o r b i o active m a ;; e r i a. 1 s . Still a not her o b je c t of this inventio n is t o propose 4 process for 'the preparation of said hy d r o p ho 11 i c po 1 y me r i c nan o particles. A flirt her object of this invention is to propose a process for the preparation of said highly monodispersed polymeric nanopart i cles disposed in aqueous buffer and free of any toxic material. A stijll further object of this invention is to propose a process for the preparation of highly monodisperssjd drug laoded polymeric nanopart icles of hydrophi11ic in nature which obviates the disadvantages associated with these of tie prior art. V e t 4 nothe r ob.j e c t o f this invention is to prepare a process for the insertion and loading oftarget drug/target substance in nanoparticles to secure them from outer intervention in vivo or cell culture invitro till they are exposed at the target site within the cell. DESCRIPTION OF INVENTION According to this invention there is provided a process for the preparation of highly monodispersed polymeric hydrophiUic nanoparticles having size of upto 100nm comprising in the steps of:- i) dissolving 0.01M-0.1M of a surfactant which is sodium bis ethyl hbxyl sulphosuccinate or AerosolOT in hydrocarbons which are alknes to obtain reverse micelles; ii) adding an aqueous solution of a monomer or preformed polymer as herein described to said reverse micelles followed by addition of a crosslinking agent and initator such as herein described; iii) subjecting the mixture to the step of polymerization; iv) drying the polymerized reaction product for removal of solvent to obtain dry nanoparticles and surfactant; v) dispersing the dry mass as obtained in step (iv) in aqueous buffer followed by treatment with calcium chloride to quantitatively remove the surfactant from the adhering nanoparticles; and vi) dialysis of the nanoparticles dispersed in aqueous buffer to remove the unreacted materials from the buffer followed by lyophilisation in a conventional manner. In accordance with this invention, the aqueous core of a reverse micellar droplet is used as a nanoreacator for the preparation of nanoparticles. The sizes of the particles which are formed primarily inside these droplet are larger than the size of the aqueous core of the droplets. Moreover, since the polymerisation take place in an aqueous medium, polymers with surface hydrophilic properties are obtained by this invention. Therefore, using reverse micellar method of the present invention, it is possible to prepare very small size nanoparticles with well as uptake by res is substantially minimized. It is possible because reverse micellar droplets i n w h i c h t he: po 1 y me r i c r ea c t i o ns are carried ou t a r e h i g h 1 y m o n o dispersed. The aqueous phase is regualted in such a manner so as to keep the entire mixture in an optically transparent micro emulsion phase. The range of the aqueous phase cannot be defined as thi s won 1d de pe n d on fa c to r s su c h as the monome r, surfactant or polarity of oil, and the only factor is that the system is in an optically transparent micro e m u 1 s i o n p h a s e. In accordance with the present invention, the monopar tides have a size of up to 100 nm, preferably a size of upto 10 nm to 100 nm. I n ac c o r d a n c e with t h i s i n v e n t i o n t he a qu e o u s core of a reverse mi cellar droplet is effectively used as nanoreactor to prepare ultra fine na no pa r t i c1es a n d t o e n c a psu1a t e the d rugs ( nor ma11y water soluble chemicals to maximum size upto that of 100-200 k Dal ton protein. By the process of the present i nvent i on ,, ex t r erne 1 y sma 11 par t i c 1 es o f size of greater uniformity and down to about iOnm diameter h a s b e e n a c h i e v e d. T he sur f a ct ant, so d i urn bis & t h y I i-! e x y 1 s li 1 p h o s u c c i n a t e- , o r A e r o s o 10 T ( i , e, A 0 T ) is di sso1ved i n n-hexane to prepare reverse micelles. To the AOT solution in hexane (usually u 0 3 M t o 0 . 1M o f AOT i n h e x a r •> e ) , a q u e o us s o 1 u t i o n s o f m o n o m e r o r p reformed p o 1 y m e r c r o s s 1 i n king age n t, initiator and drug are added and the polymerisation s do ne i n prese nc e of n i t rogen gas. A d d i t i o na1 amount of water may be added in order to get nanoparticles of larger size. The maximum amount of drug that can be dissolved in reverse micelles varies from drug to drug and has to be determined b y g r a d u. ally i n c r e a s i n g t h e a n * o u, n t o f drug till t h e c 1 e a r m i c r o e m u. i s i o n i s t r a n s f o r m e d i n t o t r a n s 1 u c e n t solution. All the stock solutions are prepared in p h o s p h a t e b u f f e r a n d t hi e contents swirled vigorously in order to ensure the transparency of the solution. The reaction mixture is purged with nitrogen gas. Polymerisation is done in nitrogen atmosphere, The solvent n-Hexane is then evaported out at a temperature, for example, of 35 C using rotary evaporator out at a temperature, for example, of 35 using rotary evaporator under low pressure when transparent dry mass is obtained. The material is dispersed in water and to it CaC12 solution is added during by drop till all the calcium salt of o i e t h y 1 h e- \ Reference is now made to Fig. 2 of the a c. c om pa ny i n g drawings which illustrates the flow diagram for na no par t ides using mi cr oemu 1 si on step A shows a reverse micelle Al, proposed from water in i1 m i c r oemu1s i o n. T hus, w hen a su rfactant is dissolved in oil, then the hydrophobic constitutent tails A2 would remain in contact with oil and an inner core A3 would comprise of hydrophi1 lie c o n s t i t u e n t s . W h e n w a. t e r i s a d d e d t o a s o 1 u t i o n con ta i ni n g r&ver se m i ce11e A1, of as the h y drop h i11i c const i tuent is so1u b1e i n water, wa t e r s a. 11 v a c t e d t o t he h y d r o p h i 11 i c d o main or core A3 A monomer, c rossIi n k i ng a ge n t, the r e qu i r e d d r u g a n d i. n i t i a. tor is added t o t he r ever se micelle Al. As t he a. f o r esa id c o ns t i t u s n t s are hy d r o p h i 11 i c in nature, such constituents go to the core* A3, Polymerisation is carried out in nitrogen atmosphere to form a polymer Bl and incapsulated drug as shown in step B. In- step ,B, an evaporation is effected under low pressure for removal of the solvent. Step c illustrates the dried mass to c ons i s t of Ma nopar t i c1es C1, an d surfactant C2. T he dried mass , is dissolved in phosphate buffer and then 307. CaCl2 added thereto drop by drop in step D to pr e c i p ita t e the sur f a c t a n t as calcium di e t hy1 he x y1su1f osu c c i na t e (DEHSS). Step D illustrates the nanopar tides Cl and calcium DEHSS. The solution of step D is centrifuged at step E to obtain clear namopar tides dispersed in buffer and the precipitate of CaCDEHSS)2. The cake of CaCDEHSS)2 may contain some absprbed nanopar tides which can be recovered by d i sso1vi n g t he ca k e in hexane and leaching the n b. n o p a r t i c 1 e s b y b u f f e r 2 to 3 times. The 1 e a c h ing s oIuti o ns are c o11e c t e d a1on y with solution E. Su c h a buffer solution containing nanoparticles may still con tai n cer t a in u h rea c ted o r tox i c materials which are removed by dialyzing the solution for two hours and then freeze dried. Normally 0.01 to 0.1M AOT in n-hexane is used. V i n y 1 p y r r o 1 i d o n e (V P) o r mi x t u r c? o f vinylpyrrolid o n e and polyethyleneglycolfumarate (PEGF) are used as monomers as they form water soluble hydrogels on polymerisation and are highly biocompatible. Another suitable polymer which has been used is bovine se r urn a 1 bum i n. 01 he r su i t a b 1 e wa t e r so 1 u. b 1 e h y dr oge1s a nd bi o c om pa t i be1 mat eri a1s c a n be use d for poIyme r i sa t i o n . In case o f hy d r ogels, t he c r oss1i nk i n g is done wi t h N f N me t hy1e ne bis a c r y1 am i de C MBA) whereas a1 bum i n i s c ross1i nked by g1utara1dehyde. In c a s e o f p o 1 y v i n y 1 p y r r o 1 i d i n g !:::' V P c r "D s s 1 i n k e d with M B A ? t h e a m o y. n t o f m o n o m e r u s e d is, for example, about 50 w7. of AOT, of the amount of crosslink ing agent (MBA) used is 1.27. w/w of the p o 1 y m e r u S u. c h a c o m p o s i t i o n hi as maximum shelf life and retention of drug by nanoparticles of this composition is also maximum. Loading of drug should be between 1% to 10% w/w of the polymer according to the solubility of the drug in the mi cellar system but it can be increased according to the solubility of tne drug. Tne 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. E x am p 1 e-1 s p r e par a t i o n o f a n a n t i ge rn 1 oa de d p C' 1 y v i n y 1 p y r r o 1 i d o n e n a n o p a r t i c I e s. An antigen, from Aspergilus fumigatus, has been used as a drug for encapsulation. In a 40ml of 0 . 0 3 M A 0 T si o 1 u t ion i n h e x a n e f 14 0 u 1 of f r e s hi 1 y distil 1e d pur e v i ny1py r r olido ne, 35u1 o f N,N me t hy1e ne b i s;. a r y 1 a m i c! e C 0 .4 9 m g / m 1 ), 2 0 u 1 of 17. f e r r o u s ammom. um sulphate solution, 40ul of 11.2% aqueous b o 1 u. t i o n o f yet r a m e t h y 1 e t h y 1 e n e d i a mine , 10 u 1 o f 3 % p o t a s s i u m p e r s u 1 p h a. t e as i n i t i a t o r an d 18 0 u 1 o f antigen (antigen)= 16 mg/ml) were added. The amount of excess buffer to be added in reverse micelles was governed by the desired size of the nanoparticles to be prepared. The volume of the excess buffer can be c a r r i e d f r om 2 e r o t o max i mum amou n t up to which microemulsion formation is possible and no phase se pa r a t i o n t a kes pi ac e. T ha so1u t i o n was h o m o g e n e o u. s a n d t r a n s p a r e n t. P o 1 y its e r i s a t i o n was d o n e in presence of N2 gas at 30 C for S hours in a t h e r m o s t a tic b a t h w i t h c o n t i n u o u. s stirring. The n a r i c> p a r t i c 1 e s o f p o lyvinylpyr r o 1 i d o n e containing e n c a p s u 1 a t e d d r u. g w o u 1 d be f o r m e d . The s o 1 v e n t was evaporated off in a rotary vacuum evaporator and the dry mass was resuspended in 5ml of water. Calculated amount of 307. CaC12 solution was added dr o p by d r o p t o p r e c i p i t at e ACT as calcium salt b i s e t b y 1 r i e x y s u 1 p hi o s u. c c i n a t e. The c e n t r i f u g e d a q u e o u. s solution contains nanoparticIss which was homogeneous and almost transparent. The cake of calcium DEHSS a f t e r c e n t r i f u. g a t i o n c o n t a i n s s o m e a m o u n t o f nanoparticles absorbed in it. It was dissolved in 10ml of n-'-hexane and the hex an-:-? solution was washed 2™ 3 ti mes ea ch t i me with 1ml water. T he p hase s epar a t e d cIea r a queous layer was drained ou t and was collected with the original filtrate. The total aqueous dispersion of Nanoparticles was then dialysec! (12,000 cut off membrane) for about 2 hou r s a gai ns t wa t e r a n d the d i a1yse d so1u t i on was lyophi1ised immediately to dry powder for subsequent u s e ,. "I" h e s a m p 1 e s h o u 1 d b e f r e e f r o m A 0 T, m o n o m e r , cross!inking agent and perdisulphate. Any trace amoun t of un react© d mate r i a 1s a nd su r f a c t a n t c ou1d be detected through HF'L.C. Perdisulphate was detected chemically using starch iodide solution and the presence;- of AOT was tested as follows: To an img/ml solution of dry powder, a drop of methylene blue dye was added. The solution was then ffi i x e d w i t h 1ml of n~ he x ane t ho r ou g h 1y and was k ep t for phase separation. The hexane layer was then tested spectrometrically at 580nm for the presence of the dye. Ex am p1e-11: 7 he na noparticles f rom po1ye t hy i e he g1y c o1f uma r a t e we r e prepared as foilows s 5 g o f po 1 yet hy 1 e ne? g 1 y c c 1600, 0.9 g of f uma r i c acid a nd 1.22m of hy dr oqu i none were mi x e d t o ge t he r a n d heated at 190 C for 7~B hours in a 100ml 3 necked f 1 a s k e q u i p p e d w i t h a t h e r m o mete r , r e f 1 u. x i n g c o n d e r s e r a n d a, nitrogen inlet. T' h e p r o d u c t o f w a s g r e e n i s h y e 11 o w v i s c o u. s 1 i q u id at r o o m o f t e m p e r a t u r e. In a 40ml of 0.06m ACT in n~hexane the f o 11 o w i n g c o m p o n e n t s were added. 10 0 u 1 o f polyethyleneglycol fumarate CO.lS&g/ml), lOul of freshly distilled vinyl pyrrolidone, lOul of N,N me t hy 1 e ne bis a r c y 1 am i de (0 .049 / g / ml), 1 Ou 1 o f 0.57. f e r r o u. s a m m o n i u m s u 1 p h a t a, 2 0 u 1 o f 11.27. T M E D a n d lOul or 20ul, as the case may be, of fluorescence isothiocyanate-dextran (FITC-dextran) Mol.wt. 1GKD of concentration 160mg/ml. 0--200ul of buffer depending on the size of the droplet were added. In the above solution M2 gas was passed for 30 mins and then lOul of 5"X, potassium perdisulphate was added as initiator with vigorous stirring. The r eaf t e r ? t he ni tr ogen gas wa s passed t h r ou gh t he so I u. t i o n for a no t he r six hou r s at 30 C."i" h e n a n o p a r t i c 1 e s w e r e r e c o v e r e d f r o m t h e a q u e o u. s s o 1 u t i o n f o 11 o wing the same met h o d as described earlier in the case of po1yvi ny1py r r o1i done pa r t i c1es. E X AMPLE - Ills P r e pa r a t i o n of Bovi na Se r urn A1 bum i n gu 11 e r a 1 de hy de na no pa r t i c 1 es . I n a 4 0 m 1 o f 0 . 0 6m A 0 T i n n - h e x a n e 2 0 0 u 1 b o v i n e serum albumin (lOOmg/ml) and 0-SQOul water depending o n t he s i z e of t he- m i c e 11 a r d r o p 1 e t s were added. The mixture was thorougly stirred at room temperature till a transparent microemulsion was formed. To the well stirred solution, 20ul 5a, g1ut rar d1ehyd& was added and the stirring was c. o n 11 n u e d f o r a n o t h e r h a. 1 f a n h o u r when t h e r i a n o p a r t idle s i were f o r m e d. T hi e a q u e o us s o 1 u t i o n of the na no part ides were prepared from the AQT so1u t i o n f oIlowing the met hod as described i n t he c ase o f po1yv i ny1py r r o1i done a bove. The nanopartides were characterised as follows: The entrapment efficiency of the FITC dextran dye i n p oi yvi ny1py rro1i do he na noparticles was determined as follows? The aqueous extract including the repeated washings were? collected and was made up the volume of 10ml. 500ml of the solution was filtered through 100KD membrane filter and 2ml phosphate buffer was added. The absorbance of the solution was measured at 493nm. The absorbance of the same concentration of free FITC in phosphate buffer was m e a s u red. F" r o m t h e d i f f e r e n c e in a b s o r b a n c e the entrapment efficiency was calculated and the values as show n i n the t a b1e was f ou nd t o be in the range of 39-447. irrespective of the size of the na no pa r t i c 1 es. We have s t u d i e d C10-~ 100 nm) . bize of the particles (nm) Entrapment (polyvinyl pyrrolidone) Ef f iciency C/O 21 40 26 44 31 42 34 40 52 39 96 40 The? size of the nano par tides was determined by laser light scattering measurements. Dynamic laser light scattering measurements for d ete r mi n i n g t he s i z e of ths nanoparticles were p e r f o r m e d u s i n g B r o o k h a v e n 9 0 0 0 instrument with 81200SM go n i o i n e te r. A r go n i on air cooled laser was operated at :488nm as a light source. The time dependence of the intensity autocorrelation function of the scattered intensity was derived by using 128 cha nne1 di g i ta1 c o rre 1 a t o r, Intensity c o r r e1a t i on data was processed by i..sing the method of cumulants. The translationa1 diffusion coefficient (T) of the particles dispersed in aqueous buffer was obtained from a nonlinear least square fit of t h e c o r r e 1 a t i o n c u r v e u s :i. n g the decay equation. F" r om the va 1 ue of the t r a ns 1 a t i o na 1 diffusion coe f f i c i e nt, t he avera ge of hy d r o dy nam i c d i ame t e r D h o f the sc a 11 e r i n g par t i c1es was calculated by Strokes-Einstein relationship D = kT/3 T h where k is Boltzman constant, n is the viscosity of the solvent at an absolute temperature T. The size of the drug loaded nanoparticles of polyvinylpyrrolidone, polyethyleneglycolfumarate and bovi ne ser urn a1 bum in were determined and representative spectra for each type are shown in the figure 3 (a) (i to iii) shown: (i) na no par tides made of polyethylene glycol f u ma ra te containing FITC-Dex t ran, (i i) na nopart i c1es made of po1yv i ny1py r r o1i do ne c o n t a i n i n g FIT C - D e x t r a n, (iii) nanopar t i c1es made o f bovine 5e rum a1bumin crosslinked with glutaraldehyde. Figure 3 (b) shows the variation of particle size wi t h t he c ha n ge of size o f the m i c r oemu1s i on droplets. Interestingly the size of the p o1yv i ny1py r r oIidone nano pa r t i d es in c r eases exponentially with the increase of droplet size whereas the same remain more or less constant in case of bovine serum albumin-gluteraldehyde particles. 1 n vi tr o r e1ease kinetic studies! A known amount of lyophilised nanoparticles encapsulating FITC-dextran was suspended in 10ml o f p h o s p h a t e b u. f f e r s a 1 i n e i n 5 0 m 1 p o 1 y p r o p y 1 e n e t u. b e s .T h e tubes were placed i n w a t e r b a t h m a i n t a :i. n e d a t 3 7 C . A t p r e d e? t e r m i n e d intervals a volume of 300ul taken from each tube and was passed through a 100KB filter (Millipore UFP2THK24) which retained the nanopart ides and the free dye came out in the filtrate. The dye concentration in the filtrate was de t e r m i ned s pe ct r o p ho t ome trieally. WE CLAIM; 1. A process for the preparation of highly monodispersed polymeric hydrophillic nanoparticles having size of upto l00nm comprising in the steps of:- i) dissolving 0.01M-0.1M of a surfactant which is sodium bis ethyl hexyl sulphosuccinate or AerosolOT in hydrocarbons which are alknes to obtain reverse micelles; ii) adding an aqueous solution of a monomer or preformed polymer as herein described to said reverse micelles followed by addition of a crosslinking agent and initator such as herein described; iii) subjecting the mixture to the step of polymerization; iv) drying the polymerized reaction product for removal of solvent to obtain dry nanoparticles and surfactant; v) dispersing the dry mass as obtained in step (iv) in aqueous buffer followed by treatment with calcium chloride to quantitatively remove the surfactant from the adhering nanoparticles; and vi) dialysis of the nanoparticles dispersed in aqueous buffer to remove the unreacted materials from the buffer followed by lyophilisation in a conventional manner. 2. A process as claimed in claim 1 wherein said nanoparticles have a size of l0nm to 100 nm. 3. A process as claimed in claim 1 wherein said monomers and/or preformed polymers are biocompatible and nonatk enic meterials selected from vinylpyrrolidone or mixture of vinylpyrrolidone and polyethyleneglycol fumarate, or their polymers such as polyvinylpyrrolidone or copolymer of polyvinylpyrrolidone and polyethleneglycol fumarate. 4. A process as claimed in claim 1 wherein said polymers are biocompatible but antigenic which is bovine serum albumine. 5. A process as claimed in claim 1 wherein said cross linking agent is N, N methylene-bis acrylamide (MBA) or glutaraldehyde. 6. A process as claimed in claim 1 wherein the said initiators are water soluble perdisulphate salts selected from potassium perdisulphate, potassium persulphate and ammonium perdisulphate and the activator- is tetra methyl ethylene diamine (TMED). 7. A process as claimed in claim 1 wherein said alkanes are preferably n- hexane. 8. A process for the preparation of highly monodispersed polymeric hydrophilic nanoparticles substantially as herein described and illustrated with reference to examples.

Documents:

497-del-1999-abstract.pdf

497-del-1999-claims.pdf

497-del-1999-correspondence-others.pdf

497-del-1999-correspondence-po.pdf

497-del-1999-description (complete).pdf

497-del-1999-drawings.pdf

497-del-1999-form-1.pdf

497-del-1999-form-19.pdf

497-del-1999-form-2.pdf

497-del-1999-form-26.pdf

497-del-1999-form-3.pdf

497-del-1999-petition-138.pdf