# US 11,078,460 B2 · Method for injectable delivery of a therapeutic agent into a fish embryo

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- Number: US 11,078,460 B2
- Document ID: US11078460B2
- Status: Granted
- Kind: B2
- Application number: 15/897,139
- Filed: 2018-02-14
- Published: 2018-08-23
- Granted: 2021-08-03
- Earliest claimed: 2017-02-18
- Applicants: Tracy Shawn Peterson
- Inventors: Tracy Shawn Peterson
- Web page: https://patentgrep.com/patent/US11078460B2

## CPC

- C12N 5/0604: {Whole embryos; Culture medium therefor}
- A01K 67/02: Breeding vertebrates
- A01K 67/027: New or modified breeds of vertebrates
- C12N 15/873: Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- A01K 2207/20: Animals treated with compounds which are neither proteins nor nucleic acids
- A01K 2227/40: Fish
- A61K 2123/00: Preparations for testing in vivo
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Fish embryos may be successfully vaccinated or therapeutically treated if the therapeutic agent is injected into the yolk sac. Therapeutic agents may be directly injected or released from microspheres and enter the circulation and tissues. Injection into the yolk sac, combined with the use of carriers, allows for a continued, controlled release of therapeutic agents and processing of antigens. Fish vaccination or therapeutic treatment, selecting fish embryos post fertilization at the one-cell to eyed egg stage of development, and injecting the yolk sac with carriers associated with an antigen(s) or therapeutic agent(s), may be fully automated.

## Claims

### Claim 1

A method of immunizing a fish embryo post fertilization, when the embryo is in the one-cell to the eyed egg stage state of development, wherein the method comprises:
selecting a therapeutic agent, said therapeutic agent to include at least one of keyhole limpet hemocyanin (KLH) or whole protein extract of Mycobacterium marinum;
obtaining embryo water wherein said embryo water comprises water or reverse osmosis (RO) water and wherein the embryo water comprises a salinity of 100 to 60,000 ppm;
obtaining an injection chamber, wherein said injection chamber is designed to immobilize an embryo specific to the type of fish to be injected, from the size of a flying fish embryo to a whale shark embryo;
filling the injection chamber with embryo water;
loading a means of injection comprising a needle for injection with the therapeutic agent, said needle having a diameter of 0.1 to 6000 microns;
obtaining a fish embryo that will receive the therapeutic agent, said fish embryo to be a zebrafish embryo;
placing the fish embryo into the injection chamber;
assuring proper stage of development of the fish embryo, wherein embryo is comprised of a plurality of cells, the stage of development is dependent upon the number of cells within the fish embryo, and the stage of development is identified visually or with the use of a microscope wherein the microscope has a magnification between 10× and 1000×;
puncturing the fish embryo with the needle to inject the therapeutic agent, wherein the fish embryo comprises a membrane and a yolk sac, and the needle punctures the membrane and the therapeutic agent is injected into the yolk sac; and
allowing the embryo to further develop.

### Claim 2 (depends on 1)

The method of claim 1 wherein the injection chamber is created by placing a first layer of gel, followed by the application of a second layer of gel and the insertion of a mold into the second layer of gel, so that once the mold is removed the injection chamber is created.

### Claim 3 (depends on 1)

The method of claim 1 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

### Claim 4 (depends on 3)

The method of claim 3 wherein the injection chamber is created by placing a first layer of gel, followed by the application of a second layer of gel and the insertion of a mold into the second layer of gel, so that once the mold is removed the injection chamber is created.

### Claim 5

A method of immunizing a fish embryo post fertilization, when the embryo is in the one-cell to the eyed egg stage state of development, wherein the method comprises:
selecting a therapeutic agent, said therapeutic agent to include at least one of keyhole limpet hemocyanin (KLH) or whole protein extract of Mycobacterium marinum;
preparing a carrier, wherein a carrier comprises a microsphere combined with the therapeutic agent, said microsphere to be selected from at least one of a conventional normal phase polymeric micelles, liposomes, empty core nanoparticles, solid nanoparticles, latex beads, gold beads, aluminum beads, alginate, microspheres, β-glucan, chitin, Polylactic-co-glycolic acid (PLGA), Polylactic acid (PLA), and/or Polycaprolactone, wherein the microsphere is combined with the therapeutic agent by at least one of coating, incorporation, binding, uptake by, absorption by, and/or adhering to a microsphere;
obtaining embryo water wherein said embryo water comprises water or reverse osmosis (RO) water and wherein the embryo water comprises a salinity of 100 to 60,000 ppm;
obtaining an injection chamber, wherein said injection chamber is designed to immobilize an embryo specific to the type of fish to be injected, from the size of a flying fish embryo to a whale shark embryo;
filling the injection chamber with embryo water;
loading a means of injection comprising a needle for injection with the therapeutic agent, said needle having a diameter of 0.1 to 6000 microns;
obtaining a fish embryo that will receive the therapeutic agent, said fish embryo to be a zebrafish embryo;
placing the fish embryo into the injection chamber;
assuring proper stage of development of the fish embryo, wherein embryo is comprised of a plurality of cells, the stage of development is dependent upon the number of cells within the fish embryo, and the stage of development is identified visually or with the use of a microscope wherein the microscope has a magnification between 10× and 1000×;
puncturing the fish embryo with the needle to inject the therapeutic agent, wherein the fish embryo comprises a membrane and a yolk sac, and the needle punctures the membrane and the therapeutic agent is injected into the yolk sac; and
allowing the embryo to further develop.

### Claim 6 (depends on 5)

The method of claim 5 wherein the injection chamber is created by placing a first layer of gel, followed by the application of a second layer of gel and the insertion of a mold into the second layer of gel, so that once the mold is removed the injection chamber is created.

### Claim 7 (depends on 5)

The method of claim 5 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

### Claim 8 (depends on 7)

The method of claim 7 wherein the injection chamber is created by placing a first layer of gel, followed by the application of a second layer of gel and the insertion of a mold into the second layer of gel, so that once the mold is removed the injection chamber is created.

### Claim 9 (depends on 5)

The method of claim 5 wherein, in the preparation of the carrier a covalent attachment, polylactic-co-glycolic (PLGA) microspheres, is used to facilitate the combination of the micro sphere with the therapeutic agent.

### Claim 10 (depends on 9)

The method of claim 9 wherein the injection chamber is created by placing a first layer of gel, followed by the application of a second layer of gel and the insertion of a mold into the second layer of gel, so that once the mold is removed the injection chamber is created.

### Claim 11 (depends on 9)

The method of claim 9 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

### Claim 12 (depends on 11)

The method of claim 11 wherein the injection chamber is created by placing a first layer of gel, followed by the application of a second layer of gel and the insertion of a mold into the second layer of gel, so that once the mold is removed the injection chamber is created.

### Claim 13 (depends on 2)

The method of claim 2 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

### Claim 14 (depends on 13)

The method of claim 13 wherein the injection chamber is created by placing a first layer of gel, followed by the application of a second layer of gel and the insertion of a mold into the second layer of gel, so that once the mold is removed the injection chamber is created.