# US 11,298,415 B2 · Alginate chitosan nanoformulation of OmpA—a Shigella protein subunit

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,298,415 B2
- Document ID: US11298415B2
- Status: Granted
- Kind: B2
- Application number: 16/093,237
- Filed: 2017-04-28
- Published: 2019-02-28
- Granted: 2022-04-12
- Earliest claimed: 2016-05-02
- Assignees: Indian Council of Medical Research
- Applicants: Indian Council of Medical Research
- Inventors: Hemanta Koley; Priyadarshini Mukherjee; Dhrubajyoti Nag; Ritam Sinha; Manoj Kumar Chakrabarti
- Web page: https://patentgrep.com/patent/US11298415B2

## CPC

- A61K 39/0283: {Shigella}
- A61K 9/5161: {Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin}
- A61K 47/36: Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61P 31/04: Antibacterial agents
- A61K 2039/542: {oral/gastrointestinal}
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Provided herein is a Formulation of OmpA including a) OmpA protein as active molecule obtained as a product of OmpA gene inserted in a plasmid comprising a novel set of forward and reverse primer set, and b) Alginate chitosan nanoparticles as vehicle.

## Claims

### Claim 1

An oral formulation of outer membrane protein A (OmpA) comprising essentially of:
a) OmpA protein as an active molecule, obtained as a product of an OmpA gene derived from Shigella flexneri 2a 2457T, inserted in a plasmid comprising a set of a forward and a reverse primer set; and
b) Alginate chitosan nanoparticles as a vehicle;
wherein the formulation has a particle diameter in a range of 240 nm to 700 nm;
wherein the formula is administered orally;
wherein the formulation has a mean particle size of 535.0 nm, a particle size distribution of 0.470 and a zeta potential of −20.78;
wherein the forward primer has a nucleotide sequence 5′-AAAAAGACACATATG CG ATT GCAG-3′ (SEQ ID NO: 1) and the reverse primer has a nucleotide sequence 5′-GTATCTCGAGAGCTTGCGCTGAGTTAC-3′ (SEQ ID NO: 2); and,
wherein bases “CATATG” in the forward primer represents restriction site specific for NcoI and the bases “CTCGAG” in the reverse primer represents restriction site specific for XhoI.

### Claim 2

A method of preparation of an oral formulation of an outer membrane protein A (OmpA), the method comprising the steps:
a) preparing a recombinant plasmid comprising an OmpA forward primer having a nucleotide sequence 5′-AAAAAGACACATATG CG ATT GCAG-3′ (SEQ ID NO: 1) and an OmpA reverse primer having a nucleotide sequence
5′-GTATCTCGAGAGCTTGCGCTGAGTTAC-3′ (SEQ ID NO: 2)
wherein the bases “CATATG” in the forward primer represents restriction site specific for NcoI and the bases “CTCGAG” in the reverse primer represents restriction site specific for XhoI;
b) transferring the recombinant plasmid to E. coli BL21 (DE3) for multiplication;
c) isolating OmpA protein as a product from the E. coli BL21 (DE3);
d) preparing chitosan with alginate nanoparticles; and
e) loading OmpA protein into said chitosan with alginate nanoparticles;
wherein the formulation has a particle diameter in a range of 240 nm to 700 nm;
wherein the OmpA is derived from Shigella flexneri 2a 2457T;
wherein the formulation is configured for an oral administration; and
wherein the formulation has a mena particle size of 535.9 nm, a particle size distribution of 0.470 and a zeta potential of −20.78.

### Claim 3 (depends on 2)

The method as claimed in claim 2, wherein preparing the plasmid is performed by common restriction sites specific to restriction enzymes NcoI and XhoI.

### Claim 4 (depends on 2)

The method as claimed in claim 2, wherein preparing the plasmid is performed through ligation of the plasmid and said primers by mixing the plasmid and primers in a molar ratio of 1:3.

### Claim 5 (depends on 2)

The method as claimed in claim 2, wherein isolating OmpA protein as a product from the E. coli BL21 (DE3) is performed in the mid log phase of said bacteria.

### Claim 6 (depends on 2)

The method as claimed in claim 2, wherein preparing chitosan nanoparticles comprises the steps of:
a) preparing a chitosan solution by dissolving chitosan in 1% (v/v) acetic acid solution at a concentration of 2 mg/ml;
b) preparing an anionic tripolyphosphate (TPP) solution by dissolving TPP in distilled water at a concentration of 1 mg/ml;
c) adding the TPP solution into the chitosan solution dropwise at 1:2 ratio;
d) forming chitosan colloid nanoparticles under mild agitation in room temperature condition;
e) formulating colloid chitosan nanoparticles at room temperature during centrifugation at 35,000 rpm for 1 hr.;
f) discarding supernatant and re-dispersing deposit in distilled water to obtain chitosan nanoparticles.

### Claim 7 (depends on 2)

The method as claimed in claim 2, wherein loading the OmpA protein into said chitosan with alginate nanoparticles comprises the steps of:
a) redispersing a colloid of said chitosan nanoparticles in 25 ml of distilled water at a concentration of 2 mg/mL under continuous ultrasonication to disaggregate the nanoparticles;
b) loading OmpA protein subunit into the chitosan nanoparticles by incubating OmpA protein obtained as a product from the E. coli BL21 (DE3) with said nanoparticles under mild agitation for 15 minutes at room temperature condition to make final concentration of OmpA protein 1 mg/mL;
c) adding dropwise OmpA-loaded chitosan nanoparticles suspensions with pH 5.1 into sodium alginate solution at a concentration of 1 mg/mL under mild agitation at pH 7 for 10-20 minutes;
d) centrifuging the resulting suspension at 3400 rpm for 5 minutes and discarding the supernatant to obtain alginate coated chitosan nanoparticles; and
e) redispersing alginate coated chitosan nanoparticles into an aqueous solution of calcium chloride (CaCl2) at a concentration of 0.524 mmol/L to crosslink the alginate layer present on the surface of chitosan nanoparticles.