Title of Invention


Abstract 100 for encapsulating a targeted molecules/drug material comprising:-i) dissolving 0.01m to 0.1m surfactant in an oil to obtain reverse micelles;ii) adding an aqueous solution of a monomer or preformed polymer to said reverse micelles and a crosslinking agent, initiator and drug or a target substance being added therein if required;iii) subjecting such a mixture to the step of polymerization; iv) drying said polymerized reaction material for the removal of solvent so as to obtain dry nanoparticles and surfactant,v) dispersing the dry mass in the aqueous buffer and vi) separating the suifactant and other toxic materials therefrom.
This invention relatos to a process for the preparation of highly ;nonodispersed polymeric hydrophilic nanoparticlos with or without target molecules encapsulated therein and having sizes of upto 100 nm and a high monodispersity. PRIOR ART
Following an administration of a drug in a living system the active substance is distributed throughout the body st a function of its physicochemical properties** and molecular structure. The final amount of drug reaching its target site may only be a small fraction of the administered dose. Accumulation of drug at the non-targeted site may lead to adverse effect and undesirable side reactions. Therefore, targeting of drug to specific body sites is necessary.
One way of modifying the biodistribution of drugs in the body is to entrap them in ultrafine drug carriers. Among these carriers, liposomes, nanoparticles and pharmacosomes have been extensively studied. The* use of liposomes as drug targeting agents is found to be limited due mainly to the problems of low entrapment efficiency, drug instability, rapid drug leakage and poor storage stability. With the turn of overcoming these
problems, the production of polymeric nanoparticles has been investigated since the last two decades. Nanoparticles are defined as solid colloidal particles ranging in «ize from about lOntr to lOOOnm.
A large number of studies hava been reported on recent advances in drug targeting possibilities and sustain release action with nanoparticles encapsulating drugs. In vivo studies have also been reported with special attention to the reticuloendothelial system (RES). Some in vivo studies concerning nanoparticles administration by oral and ocular routes have also been reported in the literature with rwspect to the possible improvements of bioavailability. These polymeric nanoparticles should be non antigenic, biocompatible and biodegradable.
The important characteristics of the particles used for targeting at specific body sites were found to be influenced mainly by two factors: (i) the size of the nanoparticles and (ii) the surface characteristics of the nanoparticles.
Parti Ices smaller than 7 nm and specially nanopartides are not filtered in the lung and their biodistribution nanoparticles, uncoatt*d and coated with poloxamer surfactant by bone marrow. These small particles in the blood serum do not adsorb serum protein through opsonisation and as a result, their circulation time in blood is considerably increased. Hydrophobic particles are removed from the circulation very ra-pidly due to opsonisation. Nanometer sized particles with hydrophilic surface remain in blood for lunger period of time so that targeting at specific sites may be facilitated.
At present, nanoparticles for drug encapsulation are prepared by methods involving either polymerisation of dispersed monomers or a dispersion of prefor-nad polymers in emulsion in presence of desired drug. The methods known in the art for the preparation of nanoparticles &re (i) dispersion polymerisation method, (ii)emulsion polymerisation method, (iii) dispersion of synthetic polymer nanospheres in emulsion, and (iv)
interfacial polymerisation technique. In all these methods emulsions of oii-in-water are used and the polymer is formed or dissolved in the oil phase.
As a result, the polyasric materials are always hydrophobic because they »re to be soluble in oil and the particles formed are nanoparticles of larger size the nanoparticles formed have broad spectrum size range and these are also highly polydispersed. Thus, in such known processes (i) one cannot prepare nanoparticles of subcolloidal size and (ii) the emulsion medium demands that the polymeric materials should be hydrophobic.
An object of this invention is to propose a novel process for ths preparation of highly monodispersed polymeric nanoparticles with or without targeted materials and having a size of upto 100 nm with a high monodispersity.
A further object of this inventon is to propose a process for the preparation of said polymeric monoparticles capable of being modulated to required sizes.
Another object of this invention is to propose a process for the preparation of said highly monodospersed polymeric nanoparticles of subcollodial size with or without targeted materials.
Still another objer.t of this invention is to propose a process for the preparation of said -hyd raphe 1 li>c polymeric nanoparticles .
A further object af this invention is to propose a process for the preparation of said highly monadispersed drug loaded polymeric nanoparticles disposed in aqueous buffer and free of any toxic material.
A still further object of this invention is to propose a process for the preparation of highly monodispersed drug loaded polymeric nanoparticles of hydrophillic in nature which obviates the disadvantages associated with these of the prior art.
Yet another object of this invention is to prepare a process for the insertion and loading of target 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 eel'..
According to this invention there is provided
a process for the preparation of highly monodispersed polymeric hydrophillic ri*noparticles of the size of upto
100 for encapsulating a targeted moiecu3 ess/drug material comprising:-i) dissolving 0.01m to. 0.1m surfactant in an oil to
obtain reverse micoll«rs»
ii) adding an aqueous solution of a monomer or
preformed polymer to said reverse micelles and a cross!inking agent, initiator and drug or a target
substance being added therein if required; iii) subjecting such a mixture to the step of
polymerization; iv) drying said polvfcieriaed reaction material for the
removal of solvent so as to obtain dry nanoparticlos and surfactant, v) dispersing tho dry mass in the aqueous buffer and
vi) separating tht? surfactant and other- toxic materials therefrom.
In accordance with this invention, the aqueous core of a revere® tnicellar droplet is used as a nanoreactor for the preparation of nanoparticles. The sizfcs of the aqueous core of such droplets &r& in "the range of Inm-lOnm. The size of the particles which Are formed primarily inside these droplets ar$ larger than the size of the aqueous core of the droplets. Moreover, since the polymerisation takes 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 hydrophilic surface so that their opsonisation as well as uptake by RES is substantially minimized. It is possible because reverse micellar droplets in which the polymeric reactions are carried out ^re highly monodispersed*
The aqueous phase is regulated 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
this would depend on factors such as the monomer, surfactant or polarity Of oil, and the only factor is that the system is in an optically transparent micro emulsion phase.
In accordance with the present invention, the monoparticles have a sige of upto 100 not,preferably a size of upto 10 nm to 100 nm.
In accordance with this invention the aqueous core of a reverse micellar droplet is effectively used as nanoreactor to prepare ultrafine nanoparticleu and to encapsulate the drugs (normally water -soluble chemicals of maximum size upto that of 100-200 k Dalton protein By the process of the present invention, extremely small particles of sise of greater uniformity and down to about iOnm diameter has been achieved.
The surfactant, sodium bis ethylhexylsulphosuccinate, or Aerosol0T (i,e,A0T) is dissolved in n-hexane to prepare reverse micelles. To the AQT solution in hexane (usually 0.03M to O.IM of ACT in hexane), aqueous solutions of monomer or preformed polymer, crosslinking agent,
initiator and drug are Added and the polymerisation is done in presence of nitrogen gas. Additional amount of water may be added in order to get nanoparticles of larger size. The maximum amount of drug that can be dissDived in reverse micelles varies from drug to drug and has to be determined by gradually increasing the amount of drug till the clear microemulsion is transformed -into translucent solution. All the stoefc solutions are prepared in phosphate buffer and the 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 evaporated out at a temperature, for example, of 35 C using rotary evaporator under low pressure when tranparent dry mass iu Obtained. The material is dispersed in water and to it CaC12 solution is added drop by drop till 3I1 the calcium salt of

diethylhexylsulphosuccinate (Ca(DEHSS)2 from AOT) is precipitated. The mixture is then subjected to centrifugation, for example, at 15000 rpm for 10 mins. The supernatant is decanted off which contains nanoparticl*m containing encapsulated drug. Some nanopartides remain absorbed in the cake of the precipitate. For complete recovery of the nanoparticles from, the precipitated calcium (DEHSS)2 the latter is dissolved in n-hexane and the nanoparticles extracted with water. The aqueous dispersion is immediately dialysed through , for example, 12,000 cut off dialysis membrane for about one hour and the liquid lypophilised to dry powder and stored at low temperature till further use.
Reference is now made to Fig.2 of the accompanying drawings which illustrates the flow diagram for nanoparticles using microemulsion step A shows a rewr&e micelle Al , proposed from water in oil microe|nulsion. Thus, when a
surfactant is dissolved in oil, then the hydrophobic constituent tails A2 would remain in contact with oil and an inner core A3 would comprise of hydrophillic constituents . When water is added to a solution containing reverse micelle Al , of as the hydrophillic constituent is soluble in water, water is attracted to the hydrophillic domain or core A3 A monomer, croBslinking-agent, the required drug and initiator is added to the reverse micelle Al. As the aforesaid constituents are hydrophillic 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 aotvent. Step C illustrates the dried mass to consist of Nanoparticles CI , and surfactant C2 . The dried mass is dissolved in phosphate buffer and then 30% CaC12 added thereto drop by drop in step D to precipitate the surfactant as calcium diethylhexylsulfosuccinate (DEHSS). Step D illustrates the nanoparticles CI and calcium DEHSS.
The solution of step D is centrifuged at step E to obtain clear nanoparticles dispersed in buffer and the precipitate of Ca(DEHSS)2.
The cake of Ca(DEHSS)2 may contain some absprbed nanoparticles Which can be recovered by dissolving the cake in hexane and leaching the nanoparticles by buffer 2 to 3 times. The leaching solutions are collected elong with solution E. Such a buffer solution containing nanoparticles may still contain certain unreacted or toxic materials which are removed by dielyzing the solution for two hours and then freeze dried.
Normally O.Oi to 0.1M AOT in n-hexane is used. Vinylpyrrolidone(VP) or mixture of vinylpyrrolidone and polyethyleneglycolfumarate(PESF) 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 serani albumin. Other suitable water soluble hydrogels and biocompatible materials can be used for polymerisation. In case of hydrogels, the crosslinking is done with N,N
methylene bis acrylamide (MBA) whereas albumin is crossl inked by glutaraldehyde. In case of polyvinylpyrroliding PVP crfcsslinked with MBA, the amount of monomer used is, for example, about 50 w% of AOT, of the amount of crosslinking agent (MBA) used is 1.2% w/w of the polymer. Such a composition has maximum shelf life and retention of drug by nanoparticles of this composition i.s also maximum. Loading of drug should be between 17. to 10% w/w of the polymer according to the solubility of the drug in the micellar system but it can be increased according to the solubility of the drug.
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-Ispreparation of an antigen loaded polyvinylpyrrolidone nanoparticles:

An antigen, from Avspergilus fumigatus, has been used as a drug for encapsulation. In a 40ml of 0.03M AOT solution in htsxane, 140ul of freshly distilled pure vinylpyrfolidone, 35ul of N,N' methylene bis arylamidts (0.49mg/ml), 20ul of i/i ferrous ammonium suplphate solution, 40ul of 11.2/C aqueous solution of yeiramethylethylenediamine (TMED), lOul of 5%. potassium persulphate as initiator and 180 ul of 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 carried from zero to maximum amount up to which microemulsion formation is possible and no phase separation takes place. The solution was homogeneous and transparent. Polymerisation was done in presence of N2 gas at 30 C for 8 hours in a thermostatic bath with continuous stirring. The nanoparticles of polyvinylpyrrolidone containing encapsulated
drug would be formed. The solvent was evaporated off in a rotary vacuum evaporator and the dry mass was resuspendd in 5ml of water. Calculated amount of 307. CaC12 solution was added drop by drop to precipitate AOT ay. calcium salt bisethylhexysulphosuccinate. The centrifuged aqueous solution contains nanoparticles which was homogeneous and almost transparent. The cake of calcium DEHSS after centrifugation contains some amount of nanoparticles absorbed in it. It was dissolved in 10ml of n-hexana and the hexane solution was washed 2-3 timfes each time with 1ml water. The phase separated clear aqueous layer was drained out and was collected with the original filtrate. The total aqueous dispersion of Nanoparticles was then dialysed (12,000 cut off membrane) for about 2 hours against water and the dialysed solution was lyophilised immediately to dry powder for subsequent use. The sample should
be free from AOT, mohower, crosslinking agent and perdisulphate. Any trace amount of unreacted materials and surfactant could be detected through HPLC. Perdisulphate was detected chemically using starch iodide solution and the presence of AOT was tested as follows:
To an lmg/ml solution' of dry powder, a drop of methylene blue dye was added. The solution was then mixed with 1ml of n-hexane thoroughly and was kept for phase separation. The hexane layer was then tested spectrophotomtetrically at 580nm for the presence of the dye.
Example-IIs The nanoparticles from polyethyleneglycolfumarate were prepared as follows:
5g of polyethylene glyco1600, 0.9-g of fumaric acid and 1.22m of hydroquinonfc were mixed together and heated at 190 C for 7-8 hours in a 100ml 3necked flask equipped with a thermometer, refluxing conderser and a nitrogen inlet . The product of was greenish yellow viscous liquid at room of temperature.
In a 40ml of 0.06m AOT in n-hexane the following components were added. lOOul of polyethyleneglycol fumarate (0.1B6g/ml), lOul of freshly distilled vinylpyrrolidone, lOul of N,N' methylene bis arcylamide (0.049/g/ml), lOul of 0.5% ferrous ammonium sulphate, 20ul of 11.27. TMED and lOul or 20ul, as the case may be, of fluorescence isothiocyanate-dextran (FITC-dextran)Mol.wt. 16KD of concentration 160mg/ml. O-200ul of buffer depending on the size of the droplet were added.
In the above solution N2 gas was passed for 30 mins and then lOul of 5% potassium perdisulphate was added as initiator with vigorous stirring. Thereafter, the nitrog&n gas was passed through the solution for another six hours at 30 C.
The nanoparticles were recoverd from the aqueous solution follcwing the same method as described earlier in the case of polyvinylpyrrolidone particles.
Example-Ills Preparation of Bovine Serum Albumin-gluteraldehyde nanoparticles.
In a 40 ml of 0.06m AOT in n-hexane 200ul bovine serum albumin (1OOmg/ml) and 0-600ul water
depending on the size of the micellar droplets were added. The mixture was thoroughly stirred at room temperature till a trasnparent microemulsion was formed. To the wall stirred solution, 20ul 5'/. glutaradlehyde was added and the stirring was continued for another half an hour when the nanoparticles were formed. The aqueous solution of the nanoparticles were prepared from the AOT solution following the method as described in the case of polyvinylpyrrolidone above.
The nanoparticles were characterised as follows:
The entrapment efficiency of the FITC-dextran dye in polyvinylpyrrolidone nanoparticles 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 measured. From the
difference in absorbances the entrapment efficiency
was calculated and the values as shown in the
table was found to bs in the range of 39-44%
irrespective of the size of the nanoparticles. We
have studied (10-100nm).
Size of the particles(nm) Entrapment
(polyvinylpyrrolidone) Efficiency(%)
21 40
26 44
31 42
34 40
52 39
96 40
The size of the nanoparticles was determined by laser light scattering measurements. Dynamic laser light scattering measurements for determining the size of the nanoparticles were performed using Brookhaven 9000 instrument with BI200SM goniometer. Argon ion 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 channel digital correlator. Intensity correlation data was processed by using the method of cumulants. The translational diffusion
coefficient (T) of the particles dispersed in
aqueous buffer was obtained from a nonlinear least
square fit of the correlation curve using the decay
equation. From the value of the translational
diffusion coefficient, the average of
hydrodynamic diameter Dh of the scattering particles
was calculated by Stokes-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 bovine serum albumin were determined and
representative spectra for each type are shown in
the figure 3(a). Fig.3(a) (i to iii) show
(i) nanoparticles made of polyethylene glycol
fumarate containing FITO-Dextran,
(ii) nanoparticles made of polyvinylpyrrolidone
containing FITC-Dextran,
(iii) nanoparticles made of bovine serum albumin
crosslinked with glutarnldehyde.
Figure 3(b) shows the variation of particle size with the change of fcize of the microemulsion droplets. Interestingly the size of the polyvinylpyrrolidone nanoparticles increases exponentially with the increase of droplet size whereas the same remain more or less constant in case of bovine sarum albumin-gluteraldehyde particles.
In vitro release kinetic studies: A known amount of lyophilised nanoparticles encapsulating FITC-dtjxtran was suspended in 10ml of phosphate buffer saline in 50ml polypropylene tubes. The tubes were placed in water bath maintained at 37 C. At predetermined intervals a volume of 500ul taken from each tube and was passed through a 100KD filter(Mlllipore UFP2THK24) which retained the nanoparticles and the free dye came out in the filtrcte..The dye concentration in the filtrate was determined spectrophotometrically.
The results are shown in figure 4, which illustrates the release of FITC-dextran dye from polyethylene glycol funtarate particles of different drug loading, and whare curve XI shown a 6.47. loading and curve X2 is 3.2"/. loading of drug.
In vivo antibody response in mice serum by injecting antigen encapsulated nanoparticles. Mice were injected subcutaneously three times at an interval of 7 days with PVP nanoparticles containing 300ug of Asporgillius fumigatus antigen of suspended in lOOul of normal saline. Each group contains five animals, three of which received the antigen entrapped in nanoparticles, one received free antigen (300ug) and the controlled received empty nanoparticles suspended in normal sline. Mice were bled at predetermined intervals and the amount of Aspergilus fumigatus specific antibody in the mice serum was assayed using indirect ELISA assay. The results are shown in figure 3, which illustrates in vivo.
Specific antibody response of antigen of Apergilus fumigatus entrapped in polyvinylpyrrolidone nanoparticles at different amount of cross linking agent with curves X3,X4, X5 and X6 having 07., 0.3%, 0.67. and 1.27. of cross linking agent.

we CLAIM.:
1. A process for the preparation of hiqhly
monodispersed polymeric fiydrophi 11 ic nanopar tides of
the eize of upto 100 for encapsulating a tarqeied
molecules/drug material comprising:-i) dissolving 0.01m to 0.1m surfactant in an oil Id
obtain reverse micellest ii) addinq an equeci solution of a monomer or
preformed polymer to said reverse micelles and a crosslinking agent, initiator and drug or a target
substance being added therein if required; iii) subjecting such a mixture to the step of
polymerization iv) drying said pol ymcr ized reaction material for the
removal of solvent so 45 to obtain dry nanopartides and sur factant, v) dispersing the drv mass in the aqueous buffer and
vi ) separating the surfactant and other toxic materials therefrom.
2. A process as claimed in claim 1 wherein said
nanoparticles have a sing of 10nm to 100nm.
3. A process as claimed in claim .1 wherein said monomers end/or prerforwed polymers are biocompatible and
nonantigenic materials such as vinylpyrrolidona or
mixture of vinylpyrrolidone and
polyethyleneqlycofumarate, or their polymers such as polyvinylpyrrolidone or copolymer of
polyvinylpyrrolidone and polyethleneglycolfumarate.
4. A process as claimed in claim 1 wherein said
polymers &re biocompatible but antigenic such as bovine
serum albumine.
5. A process an claimed in claim J. wherein said
cross linking agent is N,N methylene-bis acrylamide
(MBA) or glutar alduitvde.
6. A process as claimed in claim 1 wherein the said
initiators are water soluble perdisulphate salts like
ammonium perdisulphate and the activator is tetra methyl ethylene diamine (TNEP).
7. A process as claimed in claim 1 wherein 1%. to 10%
target substance by weight of the polymeric material is
encapsulated into to said nanoparticles.
S. A process as claimed in claim i wherein the dried nanoparticles and surfactant after removing hydrocarbon
solvent sre dispersed in buffer solution and then treated with calcium chloride to quantitatively remove
the surfactant from the adhering nanoparticles.
9. A process as claimed in claim 1 wherein the
nanoparticles dispersed in aqueous buffer is dialysed to
remove the unreacted materials from the buffer.
10. A process -as claimed in claim 1 wherien the
dispersed nanopartciles after dialysis is lyophilised and preserved.
11. A process far the preparation of highly monodispersed polymeric hydrophilic nanoparticles substantially as herein described.






2350-del-1996-description (complete).pdf








Patent Number 188751
Indian Patent Application Number 2350/DEL/1996
PG Journal Number 15/2010
Publication Date 09-Apr-2010
Grant Date 08-Aug-2003
Date of Filing 29-Oct-1996
Name of Patentee THE SECRETARY,
# Inventor's Name Inventor's Address
PCT International Classification Number C08F 110/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA