FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"NANOPARTICLES FOR DELIVERY OF BIOLOGICALLY ACTIVE MOLECULES"
Serum Institute of India Ltd., an Institute organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

STATE OF THE ART
The delivery systems for releasing biologically active
agents form a field of continuous research and development. It is known that the administration of active ingredients to the animal or human body by different administration routes has difficulties. Some peptides, proteins and polysaccharides, are not effectively absorbed through mucous surfaces given the limited permeability of epithelial barriers.This makes it necessary to develop administration systems allowing a better absorption by alternative routes to subcutaneous administration. The incorporation of active ingredients in small-sized particles is emphasized among the recently proposed possibilities to overcome the biological barriers faced by drugs.
Mucosal vaccines offer great potential since they can be administered via oral or intranasal delivery route which does not require trained personnel, avoids the use of needles and improves overall patient compliance and acceptance.
Polymeric nanoparticles have been widely investigated as carriers for drug delivery (Biomaterials 2002; 23:3193-3201} . Much attention has been given to the nanoparticles made of synthetic biodegradable polymers such as poly-.epsilon.-caprolactone and polylactide due to their good

biocompatibility (J. Drug Delivery 2000; 7:215-232; Eur. J. Pharm. Biopharm. 1995; 41:19-25). However, these nanoparticles are not ideal carriers for hydrophilic drugs because of their hydrophobic property.
Chitosan (CH) is a naturally occurring polysaccharide that is attractive for biological applications due to low immunogenicity and low toxicity (Kumar, 2000}.Chitosan is a cationic polysaccharide derived from chitin, which is a copolymer of glucosamine and N-acetyl glucosamine units (Mi et al., 1999; Gupta and Ravi Kumar, 2001; Kumar, 2000). Chitosans have been evaluated as carriers for drugs in nanoparticles in view of their biocompatilibity and biodegradability (Bayomi et al., 1998; Genta et al., 1998; Ko et al., 2003; Katas and Alpar, 2006). It was reported that CS is non-toxic and soft-tissue compatible (Biomacromolecules 2004; 5:1917-1925; Biomacromolecules 2004; 5:828-833). Additionally, it is known that CS has a special feature of adhering to the mucosal surface and transiently opening the tight junctions between epithelial cells (Pharm. Res. 1994; 11:1358-1361).
Van der Lubben et al. reported that chitosan and its derivatives are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophilic

macromolecules such as protein and peptide drugs and vaccines (Euro J Pharma Sci 2001; 14:201-207)
Chitosan nanoparticles for administering active ingredients as well as a process for obtaining them have been described in (J. Appl. Polym. Sci. 1997, 63, 125-132; Pharm. Res. 14, 1997b, 1431-6; Pharm. Res. 16, 1991a, 1576-81; S.T.P. Pharm. Sci. 9, 1999b, 429-36, Pharm. Res. 16, 1999, 1830-5; J. Control Release 74, 2001, 317-23 and U.S. Pat. No. 5,843,509). The drawback of these nanoparticles is their limited stability in certain pH and ionic strength conditions.
Chitosan NP prepared by ionotropic gelation technique was first reported by Calvo et al. , (1997b) and has been widely examined and developed (Janes et al., 2001; Pan et al., 2002). The mechanism of chitosan NP formation is based on electrostatic interaction between amine group of chitosan and negatively charge group of polyanion such as tripolyphosphate(TPP) (Bodmeier et al., 1989; Xu and Du, 2003) . This technique offers a simple and mild preparation method in the aqueous environment.
Mallaredy Vandana et al discusses chitosan nanoparticles encapsulating BSA , prepared by utilizing ionic gelation,TPP & sucrose based lyophilization ,please refer

"Optimization of physicochemical parameters influencing the fabrication of protein-loaded chitosan nanoparticles ",Nanomedicine October 2009, Vol. 4, No. 7, Pages 773-785 .
US 2008/0095810 discloses preparation of chitosan-PEG nanoparticles encapsulating DT or TT for nasal/sublingual/oral delivery.The said nanoparticles were prepared by utilizing ionic-gelation, TPP & glucose based lyophilization.The objective of this work was to study the physicochemical and in vitro release properties of chitosan nanoparticles with different molecular weights (low, medium and high) using bovine serum albumin (BSA) as a model protein for developing nanoparticle formulations that were stable and reproducible after lyophilization.
The above cited prior art discusses encapsulation of antigens in chitosan nanoparticles.Encapsulation requires measurement of entrapment efficiency and estimation of release kinetics.Also prior art discusses preparation of chitosan nanoparticles with simultaneous encapsulation of antigens, followed by lyophilization.
The inventors of the instant invention have surprisingly found a novel, unique nanoparticle system for intranasal sublingual and injectable delivery of a "biologically active molecule",wherein the chitosan nanoparticles

prepared are initially lyophilized followed by adsorption of "biologically active molecule" on said lyophilized chitosan nanoparticles resulting in maximum adsorption of antigens on lyophilized chitosan nanoparticles.
SUMMARY OF THE INVENTION
The present invention provides novel methods for preparing antigen adsorbed chitosan nanoparticle compositions having following advantageous features over existing prior art:
a) 2 to 5 times more immunogenicity as compared to existing injectable vaccine ,with respect to IgG levels and animal challenge assays;
b) applicable for delivery of any biologically active molecule,particularly for vaccine antigens;and
c) use of lyophilized chitosan nanoparticles for
adsorption of antigens, wherein chitosan is
concentrated,thus resulting in maximum antigen adsorption
efficiency.
An important aspect of the invention is a process for obtaining "antigen adsorbed chitosan nanoparticles" as defined, comprising
a) preparing an aqueous chitosan solution having pH between 4.5 and 5;

b) preparing an aqueous crosslinking agent solution of
tripolyphosphate having pH between 5.5 and 6;
c) mixing the solutions of steps a) and b) in a specific ratio, such that chitosan nanoparticles are spontaneously obtained by means of ionic gelation;
d) lyophilizing the chitosan nanoparticles in presence of a cryoprotectant for concentrating chitosan;
e) incubating mixture of lyophilized chitosan
nanoparticles having pH from 4.5 to 5.5 and biologically
active molecules having pH from 5 to 6
f) adjusting pH between 5.5 to 6 for optimal antigen
adsorption;
g) addition of a polymer surfactant to form gel;
h)nasally or sublingually administering the gel based formulation; and
characterized in that loading efficiency of said biologically active molecule is atleast 50%.
One aspect of the present invention is that the concentration of chitosan or derivative is between 0.2%w/v and 0.4 %w/v.

A second aspect of instant invention is that the concentration of tripolyphosphate(TPP) utilized during preparation of nanoparticles is between 0.1%w/v to about 0.14% w/v.
Another aspect of the instant invention is that said chitosan nanoparticles can be prepared by a method selected from ionotropic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte complex.
Also according to the present invention , Chitosan/TPP ratio can be selected from 2:1 ,3:1 & 4:1. Preferably the ratio can be 3:1.
For long term stability of formulation the chitosan nanoparticles can be lyophilized by utilizing lyoprotectants selected from but not limited to sucrose, trehalose, lactose, mannitol, and raffinose.In a preferred embodiment, the lyoprotectant can be trehalose at a concentration of between about 2.5 %(w/v) to 3% (w/v).
According to the instant invention the . said polymer surfactant is a triblock copolymer selected from one or more of pluronic F127, F88, P123 and P65.
Further according to the instant invention, the concentration of triblock copolymer can be present at a

concentration of between about 1% (w/v) to 20% (w/v).Preferably between 15%(w/v) to 20% (w/v).
Yet another aspect of instant invention is that per the instant invention the adsorbed chitosan nanoparticles have an average size of between about 400 nm to about 800 nm,particularly about 550 nm.
Also according to the instant invention, the said nanoparticle based composition can have electrical charge from +0.1mV to +50mV.
Further embodiment of present invention is that pluronic F 127 is added to antigen adsorbed chitosan nanoparticles at a concentration of 17% w/v and at gelation temperature of 35° C to form gel.
Further yet another embodiment of the current invention is that said composition can contain chitosan in a microparticle or non-microparticle form.
The composition according of the instant invention can be adapted for buccal, sublingual or nasal administration.Preferably for mucosal delivery of antigens.
DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1: Chromatograms of various concentrations of Tetanus Toxoid at pH 6.72
Figure 2: Chromatograms of Tetanus Toxoid(50 Lf/ml) at pH 2.02 to 3.0.1
Figure 3: Chromatograms of Tetanus Toxoid(50 Lf/ml) at pH
3.21 to 4.00
Figure 4: Chromatograms of Tetanus Toxoid{50 Lf/ml) at pH
4.22 to 5.01
Figure 5: Chromatograms of Tetanus Toxoid(50 Lf/ml) at pH 5.24 to 6.72
Figure 6: Chromatograms of various concentration of Diphtheria toxoid at pH 6.6
Figure 7: Chromatograms of Diphtheria toxoid (40 Lf/ml) at pH 2.0 to 3.0
Figure 8: Chromatograms of Diphtheria toxoid (40 Lf/ml) at pH 3.21 to 4.03
Figure 9: Chromatograms of Diphtheria toxoid (40 Lf/ml) at pH 4.20 to 5.03
Figure 10 : Chromatograms of Diphtheria toxoid (40 Lf/ml) at pH 5.20 to 6.0
Figure 11:Particle size of blank chitosan nanoparticle (CsNP) pre-lyophilization
Figure 12: Particle size of Blank CsNP post-lyophilization
Figure 13: Particle size of TT loaded CsNP

Figure 14: Particle size of DT loaded CsNP
Figure 15: Zeta potential of Blank CsNP before lyophilization
Figure 16 :Zeta potential of Blank CsNP after Lyophilization
Figure 17: Zeta potential of TT loaded CsNP
Figure 18: Zeta potential of DT loaded CsNP
Figure 19 : TEM of Blank CsNP
Figure 20: TEM of TT loaded CsNP
Figure 21:Calibration curve of BSA for Protein estimation
Figure 22: IgG level of controls and TT loaded CsNP
DETAILED DESCRIPTION
Definitions
The term "biologically active molecule" relates to any substance which is used to treat, cure, prevent or diagnose a disease or which is used to improve the physical and mental wellbeing of humans and animals. These biologically active molecules can include from low molecular weight drugs to molecules of the type of polysaccharides, proteins, polysaccharide protein conjugates,recombinant

proteins,peptides, lipids, oligonucleotides and nucleic acids and combinations thereof. Examples of molecules associated to these nanoparticles include proteins such as tetanus toxoid and diphtheria toxoid, polysaccharides such as heparin, peptides such as insulin, as well as plasmids encoding several proteins. In a preferred embodiment, the biologically active molecule is the diphtheria or tetanus toxoid.
The term "nanoparticle" is understood as a structure comprising a conjugate, result of the covalent bonding between chitosan and PEG through the chitosan amino groups, which conjugate is furthermore cross-linked by means of ionic gelation by the action of an anionic crosslinking agent. The formation of covalent bonds and the subsequent ionic crosslinking of the system generate independent and observable characteristic physical entities, the average size of which is less than 1 μm.
Chitosan is a natural polymer derived from chitin (poly-N-acetyl-D-glucosamine), in which an important part of the acetyl groups of the N have been eliminated by hydrolysis. The deacetylation degree is generally in a range comprised between 30 and 95%, preferably between 60 and 95%, which indicates that between 5 and 40% of the amino groups are acetylated. It therefore has an aminopolysaccharide

structure and a cationic character.
The chitosan used to obtain the antigen adsorbed chitosan nanoparticles of the present invention is a low molecular weight chitosan having molecular weight between 5 and 200 kDa, preferably between 20 and 50 kDa, more preferably between 20 and 30 kDa.
A chitosan derivative can also be used as an alternative to chitosan, understanding as such a chitosan in which one or more hydroxyl groups and/or one or more amino groups have been modified for the purpose of raising the solubility of chitosan or increasing the mucoadhesive character thereof. These derivatives include, among others, acetylated, alkylated or sulfonated chitosans, thiolated derivatives, as described in Roberts, Chitin Chemistry, Macmillan, 1992, 166.
When a derivative is used, it is preferably selected from O-alkyl ethers, O-acyl esters, trimethyl chitosans, chitosans modified with polyethylene glycol, etc. Other possible derivatives are salts, such as citrate, nitrate, lactate, phosphate, glutamate, etc. In any case, persons skilled in the art know how to identify the modifications which can be carried out on chitosan without affecting the commercial viability and stability of the final

formulation.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Example 1
Stability study of antigens
A) Stability study of TT
Stability study of TT and DT at acidic pH
Stability of TT/DT was determined by HPLC. TT/DT with various known concentration were analyzed on HPLC and standard curve was plotted retention time (RT) verses absorbance which helps to determine unknown concentration.
Chromatograms of various concentration of TT at pH 6.72
TT with various known concentration (100, 50, 25, 12.5, 6.25, 3.1, 1.5 Lf/ml) were analyzed on HPLC at pH 6.72.From chromatogram it was found that minimum detectable limit for TT on HPLC was 6.25 Lf/ml, at this concentration of TT peak was clearly visible and quantifiable.The peaks having concentration 3.1 Lf and 1.5 Lf/ml were also visible but not quantifiable because of predominant noise.The standard curve generated above was used for quantifying the TT bulk

in unknown samples. It was found that 50 Lf/ml concentration was suitable for acid treatment.
Chromatograms of Acid Treated TT at various pH
TT was treated with HCl for various pH from 2.02-3.01 for concentration 50Lf/ml were analyzed on HPLC .It was observed that at pH 2.02 to 3.01 the retention time (RT) of chromatograms was shifted from 7.9 to 8.2. The area under curve (AUC) was decreased as compare to control TT and hence it means that TT was degraded in this acidic pH. The concentration of TT (50Lf/ml) was treated with HCl at various pH from 2.02- 3.01 and analyzed on HPLC.
It was observed that at pH 3.21 to 4.0 the retention time (RT) of chromatograms remains same, but area under curve (AUC) was decreased as compare to control and hence it means that TT was degraded in this acidic pH.The concentration of TT (50Lf/ml) was treated with HCl at various pH from 4.22- 5.00 and analyzed on HPLC. It was observed that at pH 4.22 to 4.6 the retention time (RT) of chromatograms remains same, but area under Curve (AUC) was decreased as compare to control TT and hence it means that TT was degraded at pH from 4.22 to 4.6 in this acidic pH. But it was found that at pH range 4.80-5.00 the AUC and RT were remains same as control TT and hence it means that TT

was not degraded at this pH.
The concentration of TT (50Lf/ml) was treated with HC1 at various pH from 5.24- 6.72 and analyzed on HPLC.lt was observed that at pH 5.24 to 6.72 AUC and RT was remains same as control TT and hence it means that TT was not degraded at this pH.
Above data indicates that addition of TT at pH from 4-8 to above acidic pH retained stability of TT .Hence TT was added in chitosan nanoparticle suspension at pH 4.8 and above.
B) Stability study of DT
Chromatograms of various concentration of DT at pH 6.6
DT with various known concentration {80, 40, 20, 10, 5, 2.5, 1.25 Lf/ml) were analyzed on HPLC at pH 6.72 .
Minimum detectable limit of HPLC was 5 Lf/ml. Which concentration of DT peak was clearly visible and quantify. 2.5 Lf/ml and 1.25 Lf/ml concentration peak were also visible but at this concentration noise was also very predominant.At 19.6 minutes there was one peak which was visible in all chromatograms. That was peak of PBS buffer.The standard curve generated above could be used for

quantifying the TT bulk in unknown samples.
For acid treatment 40 Lf/ml concentrations was selected.
Chromatograms of acid treated DT at various pH
DT was treated with HC1 for various pH from 2.02-3.01 for concentration 40Lf/ml were analyzed on HPLC.
It was observed that at pH 2.00 to 3.00 the retention time (RT) of chromatograms was shifted from 8.3 to 8.4. The Area under Curve (AUC) was decreased as compare to control DT and hence it means that DT was degraded in this acidic pH.
DT was treated with HC1 for various pH from 3.20-4.03 for concentration 4 0Lf/ml were analyzed on HPLC.It was observed that at pH 3.20 to 4.03 the retention time (RT) of chromatograms remains same, but area under curve (AUC) was decreased as compare to control and hence it means that DT was degraded in this acidic pH.
DT was treated with HC1 for various pH from 4.20-5.03 for concentration 40Lf/ml were analyzed on HPLC.It was observed that at pH 4.20 to 5.03 AUC and RT was remains same as control DT and hence it means that DT was not degraded at this pH.

DT was treated with HC1 for various pH from 5.20-6.00 for concentration 4 0Lf/ml were analyzed on HPLC .It was observed that at pH 5.20 to 6.00 AUC and RT was remains same as control DT and hence it means that DT was not degraded at this pH.
Above data indicates that DT was stable or not degraded at pH from 4.01 to above acidic pH.Hence DT was added in chitosan nanoparticle suspension at pH 4.01 and above.
Example 2
Preparation Of Chitosan Nanoparticles
Chitosan nanoparticles were prepared by ionic gelation method, in which sodium tripoly phosphate was used as crosslinking agent comprising of following steps:
1. Chitosan (200mg) was weighed and dissolved in 40ml of (2 %)v/v acetic acid and then pH of said Chitosan solution was adjusted to 4.8 by addition of ION NaOH. Finally Volume made upto 50 ml.
2. sodium tripolyphosphate (26mg) was dissolved in 10ml of WFI and pH of this solution was adjusted to 5.0 by 0.1N HC1 wherein final volume was made up to 20 ml.

3. The sodium tripolyphosphate solution was added to above Chitosan solution in probe sonicator and probe sonication was carried out until (1 min at 90 amplitude) clear solution turned to the opalescent suspension. This suspension was stored at 4°C for further use.
In above method, chitosan gets cross-linked to sodium tripolyphosphate randomly resulting spontaneous generation of nanoparticles.
Example 3
Lyophilization of chitosan nanoparticles
Chitosan nanoparticle obtained from ionic gelation method was further freeze dried by following procedure:
1. The 2.5% of trehalose was added to chitosan nanoparticles (CsNP) suspension.
2. 2ml of sample was filled into sterile vials and partially stoppering was done.
3. The containers were loaded into the chamber and the probe was immersed into vial and was enabled.
4. Chitosan nanoparticles lyophilized using recipe in below given table.
Lyophilization Recipe

Chitosan nanoparticle was lyophilized by using following method
Table 1:Lyophilization recipe

PROCESS TEMP (°C) TIME (min) PRESSURE (mtorr)
Freezing - 40 60 -

_ 40 180 -
Primary - 34 30 200

- 34 480 100

- 15 300 100

- 15 60 100

+ 20 120 50
Secondary + 20 300 50
Total time 1530
Example 4
Loading Of TT / DT On Chitosan Nanoparticles
Loading of TT/DT on chitosan nanoparticles was done by incubation method.
1. Lyophilized Chitosan nanoparticles (l0mg) were
redispersed in WFI.
2. Tetanus Toxoid from 1000 Lf/ml to 200 Lf/ml Or Diphtheria Toxoid 1000 Lf/ml to 200 Lf/ml was added to Chitosan nanoparticles.pH of TT & DT was adjusted to 6.0.
3. These solutions were incubated for 24 hrs at 4°C with mild stirring.
4. Above suspension (TT/DT loaded Chitosan nanoparticles)

was ultracentrifuged (Beckman Coulter) at 25,Q00g for 40 minutes.
5. After ultracentrifugation pellet was observed at bottom, which was collected separately and redispersed in WFI.
6. The total amount of TT/DT present in the supernatant of CsNP was determined by Lowry's method and Lf estimation.
7. The loading efficiency of TT/DT in nanoparticles was determined by following formula.
Loading Efficiency =
Total amount of TT/DT added - Amount of TT/DT in
supernatant Total amount of TT/DT added
Example 5
Characterization of Chitosan Nanoparticles
A) Particle size analysis
Particle size analysis was done by Malvern Zetasizer Nano ZS for suspension of Chitosan nanoparticle (CsNP) pre- , lyophilization and post- lyophilization, TT loaded Chitosan nanoparticle and DT loaded Chitosan nanoparticle. Particle size analysis was done for suspension of blank Chitosan nanoparticle (CsNP), TT loaded Chitosan nanoparticle and DT loaded Chitosan nanoparticle.
Table 2: Particle size of nanoparticles pre-

lyophilization,post lyophilization & post loading of antigens

Nanoparticle Particle size (nm)
CsNP Lyophilization ) (before -200
CsNP Lyophilization ) (after -200
TT loaded nanoparticle chitosan -509
DT loaded nanoparticle chitosan -503
Particle size of CsNP before lyophilized and after lyophilized was found to be in same range i.e. 180-200nm, therefore it was concluded that addition of bulking agent like trehalose did not affect particle size of CsNP during lyophilization process.
TT/DT loaded CsNP showed increased particle size as compared to blank CsNP confirming antigen adsorption on CsNP.
B) Zeta potential analysis
Zeta potential analysis was done by Malvern Zetasizer Nano ZS for suspension of pre-lyophilization Chitosan nanoparticle (CsNP) , post- lyophilization Chitosan

nanoparticle (CsNP) , TT loaded Chitosan nanoparticle and DT loaded Chitosan nanoparticle.
Table 3: Zeta potential analysis

Nanoparticle Zeta potential (mv)
CsNP Lyophilization (pre-) + 38.5
CsNP Lyophilization (post-) + 38.3
TT loaded nanoparticle chitosan +31.7
DT loaded nanoparticle chitosan +32.3
Zeta potential of CsNP pre and post-lyophilization was found to be equivalent i.e. +38.5mv and +38.3 mv respectively, therefore it was concluded that the addition of bulking agent like trehalose did not affect the zeta potential of CsNP during lyophilization process.
Blank CsNP was having zeta potential around +38.5mv whereas after addition of negatively charged antigen it decreased to around +31.7mv.
TT/DT loaded CsNP showed decreased zeta potential than blank CsNP,confirming antigen adsorption on CsNP .

C) TRANSMISSION ELECTRON MICROSCOPY (TEM)
A TEM measurement of the colloidal solution of CsNP was performed on a Philips model CM200 instrument operated at an accelerating voltage of 200 kV. Samples for TEM analysis were prepared by placing drops of the CsNP on carbon-coated TEM copper grids. The mixtures were allowed to dry under the IR lamp.
Analysis of blank chitosan nanoparticles by TEM showed particle size around 200nm.Analysis of TT loaded chitosan nanoparticles by TEM showed particle size around 300nm which is comparably higher than blank nanoparticles.Blank and TT loaded chitosan nanoparticles were seen circular in shape. Blank and TT loaded CsNP were not aggregated. Particle size of TT loaded CsNP was higher than blank CsNP which confirms adsorption/loading of TT on CsNP.
D) Quantitative analysis of chitosan nanoparticle loaded
TT/DT by Lowry's Assay
The protein concentration of IEC purified FT fraction was determined by Lowry method. Bovine serum albumin was used as standard and OD readings of conjugate in comparison with

standard were taken at 660nm wavelength.
Absorbance of supernatant of TT loaded and DT loaded CsNP in comparison with standard BSA
TT/DT loaded Chitosan nanoparticles were ultracentrifuged and supernatant was analyzed for protein estimation. Different dilutions of standards were prepared to plot the calibration curve. Dilutions of supernatant of CsNP were prepared. Absorbance of supernatant of TT loaded and DT loaded CsNP in comparison with standard BSA at 660nm is given below in table.
Table 4: BSA std at 660nm

BSA Absorbance 660nm
Blank 0
10 μg 0.024
20 μg 0.042
40 μg 0.093
60 μg 0.149
8 0 μg 0.196
100
μg 0.242
Table 5: Absorbance of sample

Well ID Name Well Dil 660nm
SPL1 Native
TT A2 40 0.191

B2 20 0.234
SPL2 Supernat ant of TT C2 20 0.144
D2 10 0.208
SPL3 Native DT E2 40 0.116

F2 20 0.206
SPL4 Supernat ant of DT G2 20 0.104
H2 10 0.160
Table.6: Concentration of TT and DT

Sr.No Sample Total conc.
1 Native TT 11.52mg
2 Native DT 12.7 6mg
Total protein concentration of TT was 11.52 mg/ml and of DT
was 12.76 mg/ml. 1 mg of TT /DT was added in of chitosan
nanoparticle.
The Loading efficiency was determined by following formula
Loading Efficiency of TT/DT =
Total amount of TT/DT added - Amount of TT/DT in

Table .7: Loading efficiency of TT and DT

Sr.No Sample Total
Protein
Added Total conc.
in
supernatant % loading efficienc
y
1 TT 1 mg 0.4 62 mg 53.8
2 DT 1 mg 0.429 mg 57.1
According to Lowry's Assay the Loading efficiency for TT was 53.8% and for DT was 57.1%.
E) Limes of Flocculation (Lf) estimation
Lf estimation was carried out. The bulk DT and bulk TT diluted for Lf estimation as below
Table 8: Calculation of TT for Lf estimation

Parameter TT bulk (TTb) Set 2
Total TT added(μ1) 28 32 36
Amount of saline (ul) 972 968 964
ATS concentration 80 IU
The first floccules were observed at 32 ul concentration of TT. Hence the concentration of TT was found to be 80 Lf.
Table .9: Calculation of DT for Lf estimation

Parameter DT bulk (DTb) Set 4
Total DT added (μl) 30 34 38
Amount of saline(μl) 970 960 962
ADS concentration 90 IU
For DT and ADS were shown first flocculation at 34 pi concentration of DT. Hence the concentration of DT was found to be 90 Lf.
Table 10: Calculation of TT and DT per ml.

Samples Lf/ml
Bulk TT 2500
Bulk DT 2650
The 400 Lf of TT (160μl) and 400 Lf of DT (151.9μl) was
added to CsNP solution . After the ultra centrifugation of
this suspension, the supernatant of CsNP was taken for Lf
estimation of TT and DT.
Table 11: Calculation of TT in supernatant of CsNP for Lf

estimation

Parameter TT in Supernatant of CsNP
Sample (μ1) 225 2 50 275
Saline (μ1) 775 750 725
ATS concentration 50 IU
It shows first flocculation at 250 μ1 concentration of TT in supernatant of CsNP.
Hence the concentration of TT at this dilution was found to be 50 Lf.
Table 12: Calculation of DT in supernatant of CsNP for Lf estimation

Parameter DT in Supernatant of CsNP
Sample (μ1) 225 250 275
Saline (μ1) 775 750 725
ATS concentration 50 IU
It shows first flocculation at 250 μ1 concentration of DT in supernatant of CsNP.
Hence the concentration of DT at this dilution was found to be 50 Lf.
Loading Efficiency of TT/DT =


Table 13: Calculation of loading efficiency of TT/ DT

Total amount of Protein in Loading
Samples protein added in supernatant of efficie
CsNP (Lf/ml) CsNP Lf/ml ncy
TT 400 200 50%
DT 400 200 50%
According to the Lf estimation loading efficiency was found to be 50% for both TT and DT.
F) pH
The pH of the formulation was tested by Thermo scientific pH meter
Table 14: The pH of CsNP after addition of antigen and pluronic F127

FORMULATION DETAILS pH
CsNP before lyophilized 5.02
CsNP after lyophilized 5.23
TT loaded CsNP 5.42
TT loaded CsNP in PF127 5.62
DT loaded CsNP 5.48
DT loaded CsNP 5.60

Example 6
Characterization of Gel
Characterization of gel was done based on gelation temperature and rheological study. Rheological study of the gel was also carried out to determine visco-elastic nature of gel. Blank gel and antigen loaded gel formulations were characterized.
Gelation temperature
It was found that the gelation temperature for thermoreversible gel should be in the range of 27 C to 37 C. The gelation temperature of the thermoreversible formulation was found to be critical since gel formation occurs at 30°C at high concentration (>19% w/v) , such gel formation at room temperature lead to difficulty in manufacturing, handling and nasal administration .
Study also indicated that if the gelation temperature is higher than 38 C it may be transform into a liquid state at body temperature resulting in rapid nasal clearance of the administered gel. As the temperature of the nasal cavity is 35° C, further study were undertaken to obtain formulation of PF127 that gels below 35°C.
Table 15:
Determination of relation between PF gel concentration &

gelation temperature

PF gel <%w/v) Gelation temperature (°C)
Antigen(Without
Chitosan) + PF 16 37
gel
Antigen(Without
Chitosan) + PF 35.8
17 gel
Antigen (Without
Chitosan) + PF 18 35.0
gel
PF 127 at 16%, 17% and 18% showed desired gelation temperature to handle gel at room temperature and also easier administration into nasal cavities .
Effect of TT loaded CsNP and excipients on gelation temperature
The mechanism of gelation of poloxamer was found to be based on packaging of micelles and entanglement. The inclusion of drugs or additives interfered in micelle formation and consequently affected sol-gel transition.
Table 16: Gelation temperature of PF gels comprising of TT

CsNP

TT+CSNP+ PF gel (%w/v ) Gelation temperature (°C)
TTCsNP + PF 16 gel 35.4
TTCsNP + PF 17 gel 35.0
TTCsNP + PF 18 gel 34.4
The antigen concentration 5mg was kept constant for all gels. It was observed that a decrease in gelation temperature of 18 % gel was brought about by the addition of nanosuspension containing chitosan. Since this could have lead to difficulty in manufacturing, handling, for further studies chitosan nanosuspension PF 17 % gel was selected for formulation.
Gel Preparation
The 10 ml of TT loaded chitosan nanosuspension was mixed with 1.7 gm (17% w/v) of Pluronic 127 .Said mixture was incubated at 4°C for 24 hrs with stirring.
Example 7
Efficacy Study in Animal
Immunogenicity of TT loaded CsNP was performed on Swiss Albino mice. In this study alum adsorbed TT vaccine was administered by s.c. route and was considered as control

for comparison with nasally delivered TT loaded CsNP. Table .17: Groups description

Group Description Dose Route Day of
injectio
n Bleedin
g
day IgG in Sera (U/ml)
CsNP + TT
(Nasal
nanoparticles in polymer) 4Lf Nasal 0,14,28 42 50671.07 ± 8000
Control
TT
alum adsorbed 2 Lf s.c. 0 42 17649.18
Estimation of TT specific IgG in mice by ELISA
The immunogenicity of TT was estimated by using Alpha Diagnostic kit provided by Life Technologies Ltd. This mouse anti-TT IgG ELISA kit was used to detect and quantify TT specific IgG in mouse serum, mucosal secretions.
It was observed that nasally delivered TT loaded CsNP was showing higher IgG level (50,671U/ml) as compared to standard vaccine (17,64 9U/ml) thus confirming that TT loaded CsNP delivered by nasal route has great potential to generate IgG response which is higher than parenteral delivery. The immunopotency of obtained sera was further analyzed by Sera Neutralization Assay .

Sera Neutralization Test
The sera neutralizing capacity of animals immunized with tetanus toxoid when given by sublingual and intranasal routes was studied.
Table .18:

No.

Composition % Survival % Animals with paralysis
1 STD 0 —
2 STD 50 % 50 %
3 Nasal TT 100 % 0 %
It was observed that a standard sera having potency of 0.50 IU/ml, showed 50 % survival with signs of toxicity (paralysis). However nasal formulations showed potency significantly higher than 0.5 IU/ml, as there were no deaths and signs of paralysis. Thus nasal formulation showed significantly higher induction of anti-tetanus protective immunity and hence the trends observed in ELISA correlates well to sera neutralizing test.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

We claim,
1. A process for obtaining a system for release of biologically active molecules:
a) preparing an aqueous chitosan solution having pH
between 4.5 and 5;
b) preparing an aqueous crosslinking agent solution of
tripolyphosphate having pH between 5.5 and 6;
c) mixing the solutions of steps a) and b) in a specific ratio, such that chitosan nanoparticles are spontaneously obtained by means of ionic gelation;
d) lyophilizing the chitosan nanoparticles in presence of a cryoprotectant for concentrating chitosan;
e) incubating mixture of lyophilized chitosan
nanoparticles having pH from 4.5 to 5.5 and atleast one
type of biologically active molecule having pH from 5 to
6
f) adjusting pH between 5.5 to 6 for optimal antigen
adsorption;
g) addition of a polymer surfactant to form gel;
h)nasally or sublingually administering the gel based formulation; and

characterized in that loading efficiency of said biologically active molecule is atleast 50%.
2. The process according to claim 1, wherein the biologically active molecule is selected from polysaccharides, proteins, peptides, lipids, oligonucleotides, nucleic acids and combinations thereof.
3. The process according to claim 2, wherein the biologically active molecule is selected from among insulin, heparin, DNA plasmid, tetanus toxoid and diphtheria toxoid.

4. The process according to claim 1, wherein the average nanoparticle size is between 100 and 900 nanometers.
5. The process according to claim 1, wherein the electric charge {Z potential) has a value of from +0.1 mV to +50 mV.
6. A method of producing nanoparticles according to claim 1, wherein the chitosan or derivative thereof is at a concentration between 0.2%w/v and 0.4 %w/v.
7. A method of producing nanoparticles according to claim
6, wherein said chitosan is N-trimethyl chitosan, EDTA-
chitosan, low molecular weight chitosan, chitosan
derivatives, or combinations thereof.

8. A method of producing nanoparticles according to claim 7, wherein said chitosan has a molecular weight from 20 KDa to about 200 KDa.
9. A method of producing nanoparticles according to claim 6,wherein the chitosan derivative is selected from the group consisting of glycol chitosan, O-carboxymethyl chitosan, O-carboxyethyl chitosan, O-carboxypropyl chitosan, O-carboxybutyl chitosan, N,O-carboxymethyl chitosan, N-carboxymethyl chitosan, .N, O-sulfur chitosan, 1-deoxygalactit-l-yl- chitosan, 1- deoxygalucit-1-yl-chitosan and N, 0- ethylamine chitosan, hydroxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxyisopropyl chitosan, hydropybutyl chitosan and N-(2-hydroxyl)-propyl-3-trimethy1 ammonium chitosan chloride (HTACC).
10. A method of producing nanoparticles according to claim
1, wherein the sodium tripolyphosphate is at a
concentration between 0.1 %w/v and 0.14 %w/v .
11.A method of producing nanoparticles according to claim 1, wherein chitosan and sodium tripolyphosphate ratio is selected from 2:1 ,3:1 & 4:1.
12 .A method of producing nanoparticles according to claim 1, wherein the nanoparticles are lyophilized in the

presence of a cryoprotectant selected ' from the group consisting of glucose, sucrose and trehalose.
13. A method of producing nanoparticles according to claim 1,wherein said polymer surfactant is a triblock copolymer.
14. A method of producing nanoparticles according to claim 13, wherein the copolymer is selected from one or more of pluronic F127, F88, P123 and P65.
15. A method of producing nanoparticles according to claim 13, wherein the concentration of triblock copolymer is between l%w/v to about 20%w/v.
16. A method of producing nanoparticles according to claim
1, wherein the gelation temperature is between 30° C and
35° C.
17. A process for obtaining a system tor release of atleast
one type of bacterial toxoid comprising;
a) preparing an aqueous low molecular weight chitosan solution having concentration of 0.4% w/v and pH 4.8;
b) preparing an aqueous crosslinking agent solution of tripolyphosphate having concentration of 0.13% w/v and pH 5;

c) mixing the solutions of steps a) and b) in 3:1 ratio, followed by probe sonication such that chitosan nanoparticles are spontaneously obtained by means of ionic gelation;
d) lyophilizing the chitosan nanoparticles in presence of 2.5% w/v trehalose for concentrating chitosan;
e)incubating mixture of lyophilized chitosan nanoparticles having pH from 4.8 to 5.2 and tetanus toxoid or diphtheria toxoid at a concentration between 4 and 10 Lf having pH from 5.8 to 6.2.
f) adjusting pH between 5.8 to 6 for optimal antigen
adsorption;
g) addition of pluronic F 127 at a concentration of 17%
w/v and gelation temperature of 35° C to form gel;and
h)nasally or sublingually administering the gel based formulation.
characterized in that loading efficiency of tetanus toxoid or diphtheria toxoid is atleast 50%.
18. A method of producing nanoparticles according to claim 17, wherein said low molecular weight chitosan has a molecular weight from 20 KDa to about 100 KDa.

19. The process according to claim 17, wherein the average nanoparticle size is between 500 and 700 nanometers.
20. The process according to claim 17, wherein the electric charge (Z potential) has a value between +25 mV and +35 mV,preferably between +28 mV and +32 mV.
21. A nanoparticle prepared by the method according to any of the preceding claims.
22. The composition according to claim 1 or 17 ,comprising one or more bacterial toxoid antigens and at least one other component selected from group of whole cell pertussis (wP),acellular pertussis (aP) , IPV ,Haemophilus influenzae (Hib) and Hepatitis (Hep) B.
23. The composition according to claim 1 or 17 , adapted for buccal, sublingual, nasal or vaginal administration.
24. The composition according to claim 23 , wherein nasal administration of said antigen adsorbed nanoparticles results in significantly greater IgG titre and potency as compared to parenteral administration of alum adjuvanted antigen.