FIELD OF INVENTION
This invention relates to a process for producing organic/polymeric and inorganic/metal nanoparticles in the aqueous core of water in oil microemulsion system encapsulating or without encapsulating biactive molecules.
PRIOR ART
It is known to have a surfactant system, reported by Tan et.al in U.S Patent No. 6, 548, 264. However, the known system is associated with the disctint disadvantages requiring consumption of more chemicals i.e. surfactant, co-surfactant and acetone. This makes the system expensive and less ecofriendly.
Further, calcium phosphate nanoparticles of size around 300 nm and above have been reported in U.S Patent No. 6, 355, 271 and used as cariers and as controlled release matrices for biologically active materials. Ultra low size (i.e. particles of size less that 100 nm) inorganic nanoparticles which can be used as biologically safe gene delivery carrier and can be produced in a cost effective manner have been reported in U.S Patent No. 6, 555, 376. However, the major problem associated with these extremely low sized inorganic particles is there instability and tendency to aggregiate either in the reverse micelle stage or outside, under normal atmospheric conditions.
OBJECT OF THE INVENTION
An object of the present invention is to propose a process for producing organic/polymeric and inorganic/metal nanoparticles which is comparatively economical and ecofriendly.
Another object of the present invention is to propose a process for producing organic/polymeric and inorganic/metal nanoparticles which leads to reproducible results.
Still another object of the present invention is to propose a process for producing organic/polymeric and inorganic/metal nanoparticles which is simple.
Yet another object of the present invention is to propose a process for producing organic/polymeric and inorganic/metal nanoparticles wherein the particles produced are very stable and no agglomeration is observed atleast upto 3 years.
The inventive features of the present invention are indicated in the independent claims and advantageous features are reflected in the dependent claims.
STATEMENT OF INVENTION
According to this invention there is provided a process of producing organic/ polymeric nanoparticles in a water in oil system comprising steps of:
a. dissolving a 0.025M - 0.2M of a surfactant in an oil to obtain reverse micelles,
b. adding an aqueous solution of polymers/monomer with initiators/precursors to the
reverse micelles to obtain reverse microemulsion,
c. dissolving a co-surfactant to the reverse microemulsion of step (b) followed by
mixing with stirring, to obtain organic nanoparticles,
d. separating the nanoparticles from the reverse microemulsion and
e. dispersing the nanoparticles in water/solvent to remove unreacted metal salts,
surfactant molecules and oil if present.
Further according to this invention there is provided a process of producing inorganic/metal nanoparticles in water in oil microemulsion system comprising steps of:
a. dissolving a 0.025M-0.2M of a surfactant in an oil to obtain reverse
micelles,
b. dividing the reverse micelles into two equal parts,
c. dissolving aqueous solution of inorganic metal salts in one part of the
reverse micelles to obtain a transparent and optically clear reverse
microemulsion after being added with a co-surfactant,
d. dissolving aqueous solution of precipitating reagents in second part of the
reverse micelles to obtain a transparent and optically clear reverse
microemulsion after being added with the co-surfactant,
e. maintaining the same molar ratio of water to surfactant (Wo) in steps c
and d,
f. mixing both the reverse microemulsions of steps c and d with continuous
stirring to obtain metal/inorganic nanoparticles,
g. separating the nanoparticles from the reverse microemulsions and
h. dispersing the nanoparticles in water/solvent to remove unreacted metal salts, surfactant molecules and oil if present.
BRIEF DESCRITPION OF THE ACCOMPANYING DRAWINGS
Further objects and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawing and wherein:
Fig 1. shows :- a flow chart indicating the steps for producing organic/polymeric nanoparticles.
Fig. 2 shows:- a flow chart indicating steps for producing inorganic/metallic nanoparticles.
DETAILED DESCRIPTION OF THE INVENTION
This present invention relates to a process of producing particles of nanometer size dimensions (less than 100 mm) in the aqueous core of a water in oil microemulsion system containing a non-ionic surfactant and cosurfactant. Now reference may be made to figure 1 wherein 0.025 M-0.2 M surfactant such as Triton X-100 is added into an oil such as Cyclohexane and aqueous solution of polymers/monomer is added with initiators/ precursors for inititating the polymerization. The reverse micelles of surfactant into oil and aqueous solution in polymeric precursor are mixed which results in turbid solution, added with a cosurfactant dropwise to obtain a clear solution in which the cosurfactant is selected from Butanol, Pentanol and Hexanol. However, for the encapsulation of bioactive molecule such as DNA/ drug in the nanoparticles, the bioactive molecules are added in the aqueous solution prior to the addition of precursors.
The volume of aqueous solutions of the polymers can be varied accurately to control the size of the resultant particle. The ratio of molar concentrations of aqueous solution and surfactant (TX-100) is defined as Wo = [H20]/[surfactant], which varies from 3-20. The size of the nanoparticle varies directly with Wo.
The mixing of the cosurfantant with the reverse microemulsion obtained above is carried out with vigorous stirring and the resultant solution is kept on stirring for 2-3 hrs so as to allow particle growth and obtain nanoparticles.
The reaction volume thus obtained is treated with 5-20% of absolute Ethanol for breaking the reverse microemulsion and to separate the cyclohexane and water phases which is subjected to mild centrifugation carried out at the rate of 2000-5000 rpm. The centrifugation results in supernatant, which is discarded and precipitate, which is washed with 5-20% of ethanol. The steps of mild centrifugation upto washing with ethanol are repeated atleast thrice.
However, instead of absolute ethanol of solution of methanol: Benzene in the ratio of 1:3 can be employed. The particle after being washed with ethanol further washed with water/solvent followed by dispersion in water/solvent to obtain organic, polymeric nanoparticles. The above process is carried out at in temperature of 18-40°C.
Now, reference may be made to figure 2 which depicts the steps for the preparation of inorganic/metal nanoparticles wherein 0.025 M-0.2 M surfactant such as TritonX-100 is added with oil such as cyclohexane, 50% of which is mixed with aqueous solution of inorganic metal salts and another 50% is mixed with aqueous solution of precipitating reagents, which results in tubid reference microemulsions. The microemulsions thus obtained are added with cosurfactant such as 1-hexanol dropwise and separately to obtain a transparent and optically clear reverse microemulsion followed by vigorous stirring for 2-3 hrs. Same molar ratio of water to surfactant is maintained in both microemulsions. Thereafter all the steps are same as earlier with same Wo.
For the encapsulation of genetic or therapeutically active materials in the nanoparticles the same is added to both the microemulsions.
The present invention facilitates coating of the nanoparticle surface by adhesive polymeric compound and chemically conjugating ligand molecules for targeting the
nanoparticles to the specific cell type. The diameter of the void as well as loaded nanoparticles is in the range of 10 to 80 nm. The reverse microemulstions prepared are extremely stable without the addition of stabilizers for long time periods such as 3 yrs hence the reverse microemulsions prepared have long Shelf Life.
The term stability is referred to the aggregation behaviour of the particles in the reverse microemulsions forming big aggregates or clusters causing settling of the bigger aggregates hence decreasing the stability.
EXAMPLE 1
Preparation of Gold Nanoparticles
0.1 M TritonX-100 in cyclohexane was prepared. In 10ml of TritonX-100 solution in
cyclohexane, 180µl of 5% (w/v) HAuCU. 2H2O and 200 ul of 1-hexanol were added
to obtain reverse microemulsion 1 (RM-1).
In another 10 ml of TritonX-100 solution in cyclohexane, 180 µl of 5% (w/v) hydrazine hydrate and 200ul of 1-hexanol were added to obtain reverse microemulsions 2 (RJVI-2).
Both the reverse microemulsions, RM-1 and RM-2 had Wo values of 10 and both were optically clear solutions.
RM-2 was added to RM-1 with constant stirring for 3 hours for allowing complete particle growth.
EXAMPLE 2
Preparation of Metal Nanoparticles of Copper
In 10ml of 0.1 M TritonX-00 in cyclohexane, 180µl of 5% (w/v) of a copper salt (such as CuS04, 5H20) and 200ul of 1-hexanol were added to obtain reverse microemulsion 1 (RM-1).
In another 10 ml of 0.1 M TritonX-100 in cyclohexane, 180 ml of 5% (w/v) hydrazine hydrate and 200 µl of 1-hexanol were added to obtain reverse microemulsion 2(RM-2).
Both the reverse micelles, RM-1 and RM-2 had Wo values of 10 and both were optically clear solutions.
RM-2 was added to RM-1 with constant stirring. The solution changed color and was immediately frozen in liquid nitrogen.
EXAMPLE 3
Preparation of Metal Nanoparticles of Nickel
In 10 ml of 0.1 M TritonX-00 in cyclohexane, 180 µl of 5% (w/v) of nickel salt and 200
ul of 1-hexanol were added to obtain reverse microemulsion 1 (RM-1).
In another 10 ml of 0.1 TritonX-100 in cyclohexane, 180 ml of 5% (w/v) hydrazine hydrate and 200µl of 1-hexanol were added to obtain reverse microemulsion 2(RM-2).
Both the reverse micelles, RM-1 and RM-2 had Wo values of 10 and both were optically clear solutions.
RM-2 was added to RM-1 with constant stirring. The solution changed color and was immediately frozen in liquid nitrogen.
EXAMPLE 4
Preparation of Calcium Phosphate Nanoparticles
In 10 ml of 0.1 M TritonX-00 in cyclohexane, 28 µl of an aqueous solution of 1.36 M CaCl2, 152 µl of double distilled water and 200 µl of 1-hexanol were added to obtain reverse microemulsion 1 (RM-1).
In another 10 ml of 0.1 TritonX-100 in cyclohexane, 28 µl of Na2HP04, 20 µl of TRIS-HC1 buffer at a pH of 6.0 and 200 µl of 1-hexanol were added to obtain reverse microemulsion 2 (RM-2).
Both the reverse micelles, RM-1 and RM-2 had Wo values of 10 and both were optically clear solutions.
RM-2 was added to RM-1 with constant stirring for 3 hours for allowing complete particle growth.
EXAMPLE 5
Preparation of Magnesium Phosphate Nanoparticles.
In 10 ml of 0.1 M TritonX-100 in cyclohexane, 28 µl of an aqueous solution of 1.0 M MgCl2, 152 µl of double distilled water and 200 µl of 1-hexanol were added to obtain reverse microemulsion 1 (RM-1).
In another 10 ml of 0.1 TritonX-100 in cyclohexane, 28 ul of Na2HP04, 20 ul of TRIS-HC1 buffer at a pH of 6.0 and 200 µl of 1-hexanol were added to obtain reverse microemulsion 2 (RM-2).
Both the reverse micelles, RM-1 and RM-2 have wo values of 10 and both are optically clear solutions.
RM-2 was added to RM-1 with constant stirring for 3 hours for allowing complete particle growth.
SEPARATION OF NANOPARTICLES FROM REVERSE MICROEMULSION AND REDISPERSION IN WATER
The reverse microemulsion was broken by addition of absolute ethanol in the amount of 500 ul in a dropwise manner. The nanoparticles were collected by brief centrifugation at a slow speed of 3 X 103 r.p.m followed by washing with absolute ethanol to remove the surfactant and finally washing with absolute ethanol to remove the surfactant and finally washing with water.
The collected nanoparticles are dispersed in water, dialyzed in a 12 kD cutoff dialysis bags to remove residual small molecules like surfactants, cycloyhexane, un entrapped molecules, etc. to get a clear dispersion of the nanoparticles. Alternatively, the revere microemulsion can also be broken by the addition of 1:3 mixer of methanol: benzene in a dropwise manner.
The nanoparticles are collected by brief centrifugation of the solution thus obtained followed at a slow speed of 3 X 10 r.p.m followed by washing with 1:3 mixture of methanol: benzene to remove the surfactant and finally washing with water.
The collected nanoparticles are dispersed in water, dialyzed in a 12 kD cutoff dialysis bags to remove residual small molecules like surfactants, cycloyhexane, unentrapped molecules, etc. to get a clear dispersion of the nanoparticles EXAMPLE 6
50 ml of 0.1 M TritonX-100 was added to cyclohexane followed by addition of 70 ul of 0.01% of chitosan solution, 70µl of ammonia solution and 70 ul of 0.01 of Glutaraldehyde solution. This resulted in a turbid solution, which was mixed with 1-hexanol dropwise to obtain clear reverse microemulsion. The solution was stirred overnight for complete polymerization. 2 ml of ethanol was added to the solution so as to obtain the nanoparticles. The resultant solution was centrifuged at 2500 rpm, which resulted in supernatant which was discarded and particles which were washed with the same amount of Ethanol as used for breaking the reverse microemulsion. The step of centrifugation upto the step washing with ethanol was repeated thrice. Thereafter, the particles were washed and dispersed with water to obtain the required nanoparticles.
It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims: -

WE CLAIM:
1. A process of producing organic/ polymeric nanoparticles in a water in oil system
comprising steps of:
a. dissolving a 0.025M - 0.2M of a surfactant in an oil to obtain reverse micelles,
b. adding an aqueous solution of polymers/monomer with initiators/precursors to the
reverse micelles to obtain reverse microemulsion,
c. dissolving a co-surfactant to the reverse microemulsion of step (b) followed by
mixing with stirring, to obtain organic nanoparticles,
d. Separating the nanoparticles from the reverse microemulsion and
e. dispersing the nanoparticles in water/solvent to remove unreacted metal salts,
surfactant molecules and oil if present.
2. A process of producing inorganic/metal nanoparticles in water in oil system
comprising steps of:
a. dissolving a 0.025M-0.2M of a surfactant in an oil to obtain reverse
micelles,
b. dividing the reverse micelles into two equal parts,
c. dissolving aqueous solution of inorganic metal salts in one part of the
reverse micelles to obtain a transparent and optically clear reverse
microemulsion after being added with a co-surfactant,
d. dissolving aqueous solution of precipitating reagents in second part of the
reverse micelles to obtain a transparent and optically clear reverse
microemulsion after being added with the co-surfactant,
e. maintaining the same molar ratio of water to surfactant (Wo) in steps c
and d,
f. mixing both the reverse microemulsions of steps c and d with continuous
stirring to obtain metal/inorganic nanoparticles,
g. separating the nanoparticles from the reverse microemulsions and
h. dispersing the nanoparticles in water/solvent to remove unreacted metal salts, surfactant molecules and oil if present.
3. A process as claimed in claim 1 or 2 wherein a genetic material selected from DNA, RNA, PNA or any other therapeutically active derivative of nucleic acids may be encapsulated in the nanoparticles, as herein described.
4. A process as claimed in claim 1 or 2 wherein the surfactant is non ionic type which is TritonX-100.
5. A process as claimed in claim 1 or 2 wherein the oil used for making the reverse micelle and reverse microemulsion in hydrocarbon oil which is a closed chain hydrocarbon containing six carbon atoms (C.sub.6) such as cyclohexane.
6. A process as claimed in claim 1 or 2 wherein the co-surfactant is a short chain alcohol selected from butanol, pentanol and hexanol for stabilizing the reverse microemulsions.
7. A process as claimed in claim 1 or 2 wherein the separation of nanoparticles is carried out by precipitating with ethanol or methanol : benzene (1:3) mixture followed by centrifugation at a speed of 2000-5000 rpm and washing the same with ethanol or methanol: benzene (1 : 3) mixture.
8. A process as claimed in claim 1 or 2 wherein the nanoparticles after separating are dispersed in water or other solvent according to the requirement by agitation or by slight sonication.
9. A process as claimed in claim 1 or 2, wherein the molar ratio of water to surfactant (Wo) ranges from 3-20.