METHOD FOR INJECTABLE DELIVERY OF A THERAPEUTIC AGENT INTO A FISH EMBRYO

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

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 (Tobiko) 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 by at least one of visually, that is without magnification or with the use of a microscope wherein the microscope has a magnification between 1.5× and 100×; 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.

2. 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.

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

4. 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.

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

6. 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.

7. 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 (Tobiko) 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 by at least one of visually, that is without magnification or with the use of a microscope wherein the microscope has a magnification between 1.5× and 100×; 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.

8. 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.

9. The method of claim 7 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

10. 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.

11. The method of claim 7 wherein, in the preparation of the carrier a covalent attachment, polylactic-co-glycolic (PLGA) microspheres, is used to facilitate the combination of the microsphere with the therapeutic agent.

12. 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.

13. The method of claim 11 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

14. 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.

15. 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 antibiotics, antifungals, antigens for immunization, pharmaceuticals, biologicals, nutrients, immune system stimulants, adjuvants, and/or factors which act indirectly to enhance an immune response; 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 (Tobiko) 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; 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 4× 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.

17. The method of claim 16 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.

18. The method of claim 16 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

19. The method of claim 18 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.

20. The method of claim 17 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

21. The method of claim 20 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.

22. 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 antibiotics, antifungals, antigens for immunization, pharmaceuticals, biologicals, nutrients, immune system stimulants, adjuvants, and/or factors which act indirectly to enhance an immune response; 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), Polycaprolactone, and/or other natural and synthetic biopolymers, 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 (Tobiko) 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; 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 by at least one of visually, that is, without magnification or with the use of a microscope wherein the microscope has a magnification between 1.5× 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.

23. The method of claim 22 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.

24. The method of claim 22 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

25. The method of claim 24 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.

26. The method of claim 22 wherein, in the preparation of the carrier a covalent attachment is used to facilitate the combination of the microsphere with the therapeutic agent.

27. The method of claim 26 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.

28. The method of claim 26 wherein the stage of development of the fish embryo is at one-cell to eyed egg stage.

29. The method of claim 28 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.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0023] FIG. 1 illustrates Western blot analysis of Keyhole Limpet Hemocyanin (KLH) binding to Poly Lactic-co-Glycolic Acid (PLGA) microspheres, KLH was selected as representative antigen and its binding to PLGA microspheres was examined, samples of PLGA-COOH microspheres with covalently bound KLH were applied to SDS-PAGE, and separated material transferred to a PVDF membrane, to confirm the presence of KLH, membrane bound material was detected using a rabbit anti-KLH, followed by an anti-rabbit antibody coupled to horseradish peroxidase HRP, followed by 3,3′-Diaminobenzidine (DAB) for visualization, lane 1: molecular weight standards, lane 2: KLH 0.1 ug, lane 3: KLH 0.05 ug, lane 4, KLH 0.01 ug, lane 5: KLH PLGA bead prep #1, lane 6: KLH PLGA bead prep #2;

[0024] FIG. 2 illustrates a Western blot analysis of PLGA microspheres with bound Mycobacterium marinum (M. marinum) total protein, samples of PLGA microspheres with covalently bound M. marinum total protein extract were applied to SDS-PAGE and separated material transferred to a PVDF membrane, to confirm binding of M. marinum protein, an exemplary M. marinum protein, ESAT6, was identified using rabbit anti-ESAT6 followed by an anti-rabbit-HRP bound antibody and DAB for visualization, lane 1: molecular weight standards, lane 2: recombinant 25 ESAT6, lane 3: bead labeling elute, lane 4: M. marinum protein bead prep #1, lane 5: M. marinum protein bead prep #2;

[0025] FIG. 3 illustrates the presence of IgM antibodies generated by zebrafish immunized against Keyhole Limpet Hemocyanin (KLH) 1 month post-immunization, KLH antigen and BSA, were separated by SDS-PAGE and transferred to a PVDF membrane, the detection of zebrafish antibodies was achieved using antibodies specific for zebrafish IgM (AnaSpec, Inc., AS_55789S) coupled with HRP, followed by DAB for visualization, lanes: L, Molecular weight markers, KLH; Keyhole Limpet Hemocyanin, BSA, bovine serum albumin control protein;

[0026] FIG. 4 illustrates the presence of IgM and IgZ antibodies generated by zebrafish immunized against M. marinum, 1 month post-immunization, M. marinum total protein antigens, were separated by SDS-PAGE and transferred to PVDF membrane, the PVDF membrane was incubated with pooled (10 fish) whole fish homogenate from both immunized and non-immunized fish, the detection of zebrafish antibodies was achieved using antibodies specific for zebrafish IgM and IgZ (AnaSpec, Inc., AS_55789S) coupled with HRP, followed by DAB for visualization, lanes: 1, molecular weight markers; lanes 2, whole fish homogenate from vaccinated zebrafish;

[0027] FIG. 5 illustrates the presence of IgM antibodies generated by zebrafish immunized against KLH, 2 months post-immunization, methods are the same as for FIG. 3, the PVDF membrane was incubated with whole fish homogenate from immunized (IgM) and non-immunized fish (control) (pool of 10 each) as described in the examples;

[0028] FIG. 6 illustrates the presence of IgM antibodies generated by zebrafish immunized against KLH, 4-months post-immunization KLH antigen and BSA were separated by 4 to 12% SDS-PAGE and transferred to a PVDF membrane, methods are the same as described in FIG. 3, lanes, L, Molecular weight markers; KLH; Keyhole Limpet Hemocyanin; BSA, bovine serum albumin control protein;

[0029] FIG. 7 illustrates the absence of IgM antibodies to KLH in non-immunized zebrafish 4 months post-injection, zebrafish were prepared as in FIG. 6, however, zebrafish were injected with carriers not associated with antigen, as in FIG. 6, antigen was separated by 4 to 12% SDS-PAGE and transferred to a PVDF membrane, lane, L, Molecular weight markers;

[0030] FIG. 8 demonstrates the general procedure of injection of therapeutic agent bound microspheres into the fish embryo yolk sac;

[0031] FIG. 9 illustrates how therapeutic agent bound microspheres, once injected into the fish embryo yolk sac, are used to vaccinate fish embryos;

[0032] FIG. 10 illustrates how therapeutic agent bound microspheres, in this non-limiting example using an antibiotic, are used to treat or prevent infection in a larval fish;

[0033] FIG. 11 illustrates the method of vaccination; and

[0034] FIG. 12 illustrates the method of vaccination including the preparation of the microspheres used for vaccination.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure reveals a method of vaccinating fish embryos with a therapeutic agent 1, wherein said vaccination takes place after fertilization and in particular within the one-cell to eyed egg stage of development comprising, not necessarily in sequential order, the following steps:

[0036] Selecting an appropriate therapeutic agent 2. [0037] A therapeutic agent shall include at least one of all agents that may provide benefits to fish. By way of example and not by limitation, these include antibiotics (for example, keyhole limpet hemocyanin (KLH) or whole protein extract of Mycobacterium marinum), antifungals, antigens for immunization, pharmaceuticals, biologicals, and nutrients. Also included are agents which do not act directly to benefit fish but act in combination with other therapeutic agents, including immune system stimulants or adjuvants, which enhance an immune response in a host, after exposed to an antigen. Therapeutic agents including antigens may also be the products of genetic engineering. In addition to antigens, factors which act indirectly to enhance an immune response may also be associated with carriers. By way of example, M-cell homing peptide (amino acid sequence (CKSTHPLSC), may be associated with a carrier to create a specific targeting mechanism for larval and adult teleost fish. The term “Antibiotic” as used herein is meant to include but not be limited to florfenicol (Aquaflor® Type A and CA1), oxytetracycline (Terramycin® 200 for Fish (oxytetracycline dehydrate), Type A Medicated Article and OxyMarine™ oxytetracycline HCL Soluble Powder-343, Terramycin-343, TETROXY Aquatic), sulfamerazine, and sulfadimethoxine/ormetoprim combination (Romet-30®). Antifungal is meant to include but not be limited to triazole antifungals (fluconazole, itraconazole, voriconazole), allylamine antifungals (terbinafine), and amphotericin B. Biologicals include but not be limited to: peptides, nucleotides, nucleosides, antibodies, levamisole, interleukin-2, interleukin-7, interleukin-9, siRNA and DNA, melittin, and nutraceutical class therapeutics. Nutrients is meant to include but not be limited to L-lysine, L-arginine, xanthophyll, chitosan, glucosamine, plant proteins, micronutrients (vitamins and minerals), probiotics, and essential and non-essential amino acids including Taurine.

[0038] Obtaining embryo water 3. [0039] Embryo water is derived from available source water or a combination of reverse osmosis (RO) water and available synthetic sea salt mixture to a salinity of 100 to 60,000 ppm and is specific to the type of embryo that will receive the injection.

[0040] Obtaining an injection chamber, which also comprises a well plate, into which an embryo will be placed 4 to immobilize the embryo, wherein said injection chamber is designed to hold a fish egg specific to the type of fish to be injected, from the size of a flying fish (Tobiko) fish egg to a whale shark fish egg. [0041] During this step, it is possible to create an injection chamber, by way of example but not limited to, a first layer of gel created and allowed to solidify, followed by the application of a second layer of gel and a mold placed in the liquid so that once the mold is removed an injection chamber is created. Said gel is created from a substance such as but not limited to agarose. Once the injection chamber is created the injection chamber is filled with embryo water.

[0042] Loading an injection technique with a therapeutic agent 5. [0043] Any method of injection, including fully automated or with speed trajectory optimization of the micropipette injection motion may be utilized in the present invention provided it is able to inject the therapeutic agent or a solution containing carriers by way of example PLGA beads, into the yolk sac of a fish embryo without compromising vital structures (Chen P. C. Y., Zhou S., Lu Z. et al. Int. J. Control Autom. Syst. (2015) 13: 1233; Spaink H. P., Cui C, Wiweger M. I et al. Methods (2013) 62(3): 246-254; Wang W, Liu X, Gelinas D, Ciruna B, Sun Y (2007) PLoS ONE 2(9): e862). The injection technique is filled with the therapeutic agent, wherein the injection technique shall further comprise a needle having a diameter of 0.1 to 6000 microns.

[0044] Obtaining an embryo that will receive the therapeutic agent 6. [0045] A newly spawned embryo is selected for vaccination, most preferably within one hour of fertilization, wherein the embryo is in the one-cell to eyed egg stage of development.

[0046] Placing the embryo into the injection chamber 7.

[0047] Assuring proper stage of development of the embryo 8. [0048] The assessing the stage of development and the monitoring of the injection process is observable visually, that is, without magnification, or microscopically, wherein the magnification of the microscope is typically between 1.5× and 1000× magnification. Ideally, embryos are injected while in the single cell stage of development. However, fish embryos may be vaccinated, as long as injection is possible without injury to vital developing anatomical structures. By way of non-limiting example, immunizing zebrafish embryos that have exceeded 4 hours post-fertilization is preferably avoided.

[0049] Puncturing the embryo with the needle to inject the therapeutic agent into the embryo yolk sac 9. [0050] The embryo comprises a membrane, and yolk sac. Insertion of the needle into an embryo is performed with the use of micromanipulators while the process is monitored microscopically. Injection of a volume of therapeutic agent, non-destructively, is accomplished, by inserting the needle through a membrane and into the yolk sac, and applying pressure to the therapeutic agent in the needle to inject the volume, after which the needle is withdrawn. Effective amounts of therapeutic agent to be injected are expected to vary depending on the therapeutic agent and species of fish.

[0051] Allowing the embryo to further develop 10. [0052] The injected embryos may subsequently be allowed to develop in any suitable environment. Although it is envisioned that this procedure will typically take place at an aquaculture facility such as a fish hatchery, where injected embryos will be returned to controlled conditions for continued development, no such limitation is placed on the further treatment or development of the injected embryos.

[0053] The disclosure may also comprise an additional step that involves the preparation of a carrier 10. [0054] A carrier is microsphere combined with a therapeutic agent. A microsphere can be but is not necessarily limited to 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 Polycaprolactone (PCL) as well as other natural and synthetic biopolymers. The microsphere is combined with the therapeutic agent by at least one of coating, incorporation, binding, uptake by, absorption by, or adhering to a microsphere, including the use of linking agents that may aid in the association of either therapeutic agents or components. In some instances covalent attachment, for example, the use of polylactic-co-glycolic (PLGA) microspheres, may be useful with some carriers. For example, covalent attachment of biomolecules using water soluble carbodiimides is described by Hoffman et al., “Covalent Binding of Biomolecules to Radiation-Grafted Hydrogels on Inert Polymer Surfaces,” Trans. Am. Soc. Artif. Intern. Organs, 18, 10-18 (1972); and Ito et al., “Materials for Enhancing Cell Adhesion by Immobilization of Cell-Adhesive Peptide,” J. of Biomed. Mat. Res., 25, 1325-1337 (1991). The carrier is in a solution and it is that solution with carrier that is injected into the fish.