Formulations for oral delivery of polypeptides, antibodies and proteins and their use

Abstract

Nanoparticles comprising a composition comprising a polypeptide having a molecular weight greater than 50,000 g / mol (e.g., IgY antibody), wherein the composition is encapsulated in a material comprising a biocompatible biodegradable polymer, are provided. Also provided are methods of preparing these nanoparticles and the use of the nanoparticles as a therapeutic agent for treating a disease state.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 341,727, filed May 13, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] Technical Field The present invention relates to nanoparticles comprising a composition comprising a polypeptide (e.g., an antibody such as an IgY antibody, and a protein) having a molecular weight greater than 50,000 g / mol, wherein the composition is encapsulated in a material comprising a biocompatible biopolymer. Also provided are methods for preparing these nanoparticles and the use of the nanoparticles as therapeutic agents for treating disease states. Bioerodible

Background Art

[0003] One of the problems with oral delivery of therapeutic proteins such as IgY antibodies is that most proteins have a shorter lifespan and very little absorption in the gastrointestinal tract. Therefore, there is a need for improved formulations of therapeutic proteins such as IgY antibodies for oral delivery.

Summary of the Invention

[0004] erosiveness In certain embodiments, the present disclosure relates to nanoparticles comprising a composition comprising a polypeptide, an antibody, or a protein, wherein the composition is encapsulated in a material comprising a biocompatible biopolymer and a surfactant polymer. In certain embodiments, the biocompatible biopolymer erosiveness

[0005] The polymer is polymethacrylate. In certain embodiments, the nanoparticles have a diameter of less than 300 nm. In certain embodiments, the nanoparticles contain at least 5% w / w of polypeptide. In certain embodiments, the polypeptide is IgY. In certain embodiments, IgY is obtained from highly immunized eggs. In certain embodiments, the composition is a highly immunized egg product (product). In certain embodiments, the composition is a defatted fraction from egg yolk containing at least 95% by weight of IgY. In certain embodiments, the composition is purified IgY.

[0005] In certain embodiments, this disclosure relates to a pharmaceutical composition comprising nanoparticles and a pharmaceutically acceptable carrier as described herein. In certain embodiments, the pharmaceutically acceptable carrier is suitable for oral administration. The pharmaceutical composition may be in a form selected from the group consisting of powders, tablets, enteric-coated tablets, capsules, enteric-coated capsules, suspensions, solutions, and oral beverages. A substance forming a controlled-release coating can be used to provide an enteric coating for producing enteric-coated tablets or capsules. For example, the substance may be one or more selected from the group consisting of hydroxypropyl methylcellulose phthalate, hydroxymethyl ethylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethylcellulose, methacrylate-methyl methacrylate copolymer (e.g., EUDRAGIT® L 100 and EUDRAGIT® S1 00, Evonik), and methacrylate-ethyl acrylate copolymer (e.g., EUDRAGIT® L 100-55 and EUDRAGIT® L 30D55, Evonik).

[0006] In certain embodiments, the Disclosure relates to a method for delivering a polypeptide to a subject, comprising the step of administering to the subject by oral administration a nanoparticle containing the polypeptide described herein or a pharmaceutical composition containing nanoparticles containing the polypeptide. In certain embodiments of the aforementioned method, the polypeptide is IgY. In certain embodiments, IgY is obtained from highly immunized eggs. In certain embodiments, the composition in the nanoparticles is a highly immunized egg product. In certain embodiments, the composition in the nanoparticles is a defatted fraction from egg yolk containing at least 95% by weight of IgY. In certain embodiments, the composition contained in the nanoparticles contains purified IgY. In certain embodiments, the biocompatibility of the nanoparticles is erosiveness The polymer comprises polymethacrylate. In certain embodiments, the nanoparticles have a diameter of less than 300 nm. The particles can be used for oral delivery of polypeptides. The nanoparticles may be contained in tablets, capsules, suspensions, emulsions, or beverages for oral administration. In certain embodiments, the nanoparticles contain at least 5 wt% polypeptide based on the total weight of the nanoparticles.

[0007] In certain embodiments, the nanoparticles further comprise a surfactant polymer. The surfactant polymer can be selected from poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

[0008] The present invention also provides a method of medical treatment, which includes the step of administering an effective amount of polypeptide-containing nanoparticles to a patient.

[0009] Furthermore, a method for producing nanoparticles containing polypeptides having a molecular weight greater than 50,000 g / mol, the method comprising the steps of: mixing a solvent with the polypeptide to form a mixture; and providing the mixture to a biocompatible bio erosivenessA method is also provided which includes the steps of mixing a polymer, a surfactant polymer, and an organic solvent; emulsifying to produce an emulsion; high-pressure homogenizing the emulsion at a pressure of at least 10,000 psi; and evaporating the solvent to leave nanoparticles, wherein the surfactant polymer is used to stabilize the emulsion.

[0010] Furthermore, nanoparticles containing a composition comprising polypeptides, wherein the composition is biocompatible. erosiveness Nanoparticles are also provided, encapsulated in materials containing polymers and surfactant polymers. erosiveness The polymer may include polymethacrylate. erosiveness The polymer may comprise a methacrylic acid-methyl methacrylate copolymer (1:1) or a methacrylic acid-methyl methacrylate copolymer (1:2), or a combination thereof. The nanoparticles may have a diameter of less than 300 nm. The nanoparticles may contain or may contain at least 5 wt% polypeptide based on the total weight of the nanoparticles. The polypeptide in the nanoparticles may be IgY. IgY can be obtained from highly immunized eggs. The composition within the nanoparticles may be a highly immunized egg product. The composition within the nanoparticles may be a defatted fraction from egg yolk containing at least 95 wt% IgY. The composition within the nanoparticles may be purified IgY, or may contain it. In the nanoparticles provided herein, the surfactant polymer may be selected from poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

[0011] Also provided is a method for delivering polypeptides, antibodies, or proteins to a target, comprising the step of administering a pharmaceutical composition containing nanoparticles described herein to the target by oral administration. The polypeptide contained in the nanoparticles may be IgY. IgY can be obtained from highly immunized eggs. The nanoparticles may contain a composition containing a highly immunized egg product. The nanoparticles may contain a composition containing a defatted fraction from egg yolk containing at least 95% by weight of IgY. The nanoparticles may contain a composition containing purified IgY. In this method, the delivered nanoparticles are biocompatible. erosiveness May contain polymers. Biocompatible biological erosiveness The polymer may include polymethacrylate. erosiveness The polymer may include a methacrylic acid-methyl methacrylate copolymer (1:1) or a methacrylic acid-methyl methacrylate copolymer (1:2), or a combination thereof. The nanoparticles may have a diameter of less than 300 nm. The nanoparticles may contain at least 5% w / w of polypeptide based on the total weight of the nanoparticles.

[0012] Furthermore, a method for preparing the nanoparticles described herein is also provided. This method involves (a) polypeptide composition containing , water, and surfactant polymer - (b) preparing a first mixture containing (c) a biocompatible bio erosivenessThe method includes the steps of (d) adding a polymer to ethanol and sonicating it to produce a second mixture, (e) mixing the first and second mixtures with an organic solvent to form a third mixture, (f) homogenizing the third mixture using a probe homogenizer at a speed of about 5,000 to 15,000 rpm to produce a crude emulsion, (g) subjecting the crude emulsion to high-pressure homogenization using a high-pressure homogenizer at a pressure of about 10,000 psi to 30,000 psi to form a fourth mixture, and (g) evaporating the ethanol and organic solvent from the fourth mixture to produce a suspension containing nanoparticles. The method may also include the step of centrifuging the suspension to obtain nanoparticles. In this method, the surfactant polymer can be selected from poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof. The organic solvent may include anhydrous ethanol, dichloromethane (DCM), acetonitrile, acetone, ethyl acetate, chloroform, and combinations thereof. In some methods, the organic solvent includes dichloromethane. The first mixture may contain about 2 wt% to 5 wt% of polypeptide, which may be IgY. The second mixture contains 10 mg / mL to 20 mg / mL of biocompatible biochemical solution. erosiveness The mixture may contain polymers. In some embodiments, the surfactant polymer may contain 0.5 to 2.5 wt% poly(vinyl) alcohol (PVA), or 0.5 to 2.5 wt% polyvinylpyrrolidone (PVP), or a combination thereof. The first mixture may contain 0.75 wt% to 2 wt% PVA. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a graph showing the calibration curve for IgY quantification. [Modes for carrying out the invention]

[0014] In certain embodiments, the Disclosure relates to nanoparticles comprising a composition comprising a polypeptide, wherein the composition is biocompatible with bio-particles. erosiveness The present invention relates to polymers and nanoparticles encapsulated within surfactant polymers exhibiting at least weak surfactant activity. Examples of surfactant polymers include polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof. The applicant is concerned with biocompatible bio erosiveness We have identified an encapsulation method that demonstrates that polypeptides having a molecular weight greater than 50,000 g / mol or greater than 100,000 g / mol, such as IgY, encapsulated in nanoparticles containing a polymer (e.g., EUDRAGIT® L 100 or EUDRAGIT® S 100 (Evonik Industries AG, Essen, Germany)) or a combination thereof with a surfactant polymer, remain intact throughout the encapsulation process, resulting in reduced nanoparticle size and high drug loading of nanoparticles. In certain embodiments, the encapsulated composition is a highly immunized egg product, and the polypeptide is an IgY antibody. In some embodiments, the nanoparticles contain a composition comprising purified IgY antibody.

[0015] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. All patents, patent applications, published applications and publications, websites and other published materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise stated. If there are multiple definitions of a term herein, the definition in this section shall prevail. Where URLs or other such identifiers or addresses are referenced, it is understood that such identifiers may change and certain information on the Internet may appear and disappear, but equivalent information can be found by searching the Internet. References thereto serve as evidence that such information is available and publicly disseminated.

[0016] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0017] As used herein, all ranges include upper and lower limits. As used herein, a description of a numerical range for a variable is intended to convey that the variable can be equal to not only any value within that range but also any subrange subsumed by a broader range. Thus, a variable can be equal to any integer value or value within a numerical range, including the endpoints of the range. By way of example, a variable described as having a value between 0 and 10 can be 0, 4, 2 - 6, 2.75, 3.3 - 4.4, etc.

[0018] As used herein, "about" is an approximate term as would be understood by one of ordinary skill in the art and is intended to include minor variations in the quantity as literally recited. Such variations include, for example, standard deviations associated with techniques commonly used to measure the components or ingredients of a mixture or composite material or other properties and characteristics. All values characterized by the modifier "about" are also intended to include exactly that numerical value in combination with it. Thus, "about 5 percent" means "about 5 percent" and also "5 percent".

[0019] As used herein, the terms "comprises" and "comprising" are inclusive and open-ended and not exclusive. When used in this specification and the claims, the terms "comprises" and "comprising" and their variants mean that the specified feature, step, or component is included, but do not exclude other features, steps, or components.

[0020] Unless otherwise expressly indicated, all compositions described herein are intended to embrace compositions consisting of, consisting essentially of, and including the various components specified herein.

[0021] In this specification and the claims, the singular form includes plural referents unless the context clearly dictates otherwise. As used herein, the word "or" is used in the "inclusive" sense of "and / or" and not in the "exclusive" sense of "either / or", unless specifically indicated otherwise.

[0022] As used herein, the term "exemplary" means "serving as an example or illustration" and should not be construed as being preferred or advantageous over other configurations disclosed herein.

[0023] Unless otherwise indicated, each individual feature or embodiment herein can be combined with any other individual feature or embodiment described herein without limitation. Such combinations are specifically contemplated as being within the scope of the invention, whether or not explicitly described as combinations herein.

[0024] As used herein, the term "subject" includes members of the animal kingdom including, but not limited to, humans.

[0025] As used herein, "weight percent" or "wt%" refers to the concentration of a substance obtained by dividing the weight of the substance by the total weight of the composition and multiplying by 100.

[0026] The term "hyperimmunization" means repeated exposure to one or more antigens such that the immune response is elevated and maintained beyond the natural non-exposed state.

[0027] "Hyperimmune state" refers to an elevated immune response in an egg-laying animal that has been hyperimmunized.

[0028] The term "egg" as used herein refers to a whole egg (edible, highly immunized, or otherwise). The term "egg product" as used herein refers to a whole egg or any product (product) or fraction obtained from a whole egg. In certain embodiments, the egg product is egg yolk, for example, egg yolk powder. In other embodiments, the egg product is egg white, for example, egg white powder. In other embodiments, the egg product is obtained from a whole egg, for example, whole egg powder (e.g., spray-dried whole egg powder).

[0029] The term "control egg" refers to eggs obtained from ovipositors that are not maintained in a highly immunized state, i.e., from animals that are not highly immunized. The term "control egg product" refers to a control egg or an egg product obtained from a control egg.

[0030] The term "highly immunized eggs" refers to whole eggs obtained from ovipositors that have been maintained in a highly immunized state, i.e., ovipositors that have been highly immunized. The term "highly immunized egg products" refers to highly immunized eggs or any products obtained from highly immunized eggs.

[0031] In certain embodiments, the highly immunized egg product is a concentrate. As used herein, the term “concentrate” refers to a highly immunized egg product that has been at least partially purified such that the concentration of antibodies in the concentrate is greater than the concentration of antibodies in the highly immunized egg.

[0032] The term "egg powder" refers to dried whole eggs. In some embodiments, the egg powder is spray-dried.

[0033] The term "oviparous animal" means any oviparous animal, including any animal that lays eggs, such as birds, fish, and reptiles.

[0034] The term "birds" refers to animals that belong to the class Aves. Examples of birds include, but are not limited to, chickens, turkeys, geese, ducks, pheasants, quail, pigeons, and ostriches.

[0035] The term "administer" means any method of delivering a substance to a subject, including orally, intranasally, parenterally (intravenously, intramuscularly, or subcutaneously), rectally, topically, or intraocularly.

[0036] The term "antigen" refers to a substance that can induce humoral antibodies and / or cell-mediated immune responses, rather than immune tolerance. This term implies the ability to not only stimulate an immune response but also to react with its products, such as antibodies.

[0037] As used herein, “antibody” is a protein comprising at least one complementarity-determining region that binds to a specific target antigen. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody may comprise two heavy (H) chain variable regions and two light (L) chain variable regions. In certain embodiments, the antibody is a polyclonal antibody. In certain embodiments, the antibody is an IgY antibody.

[0038] As used herein, "IgY" refers to one or more IgY antibodies.

[0039] As used herein, the term "polyclonal antibody" refers to a group of antibody molecules capable of immune responses to different epitopes on a particular antigen.

[0040] As used herein, “nanoparticles” refers to particles or structures in the nanometer (nm) range, typically with a diameter of about 1 to about 1000 nm.

[0041] As used herein, the term “loading percentage” refers to the ratio of the weight of a polypeptide, such as an IgY antibody, to the weight of nanoparticles multiplied by 100.

[0042] As used herein, "Dalton" is a unit of molecular weight abbreviated as Da, where 1 Da is equal to 1 g / mol.

[0043] As used herein, "polypeptide" refers to a polymer comprising multiple linked amino acid residues and having a molecular weight of at least 50,000 Da (g / mol).

[0044] As used herein, "protein" refers to a polymer comprising multiple linked amino acid residues and having a molecular weight of at least 50,000 Da (g / mol).

[0045] The IgY antibody molecule has a structure similar to that of the IgG antibody, with each having two heavy chains (H) having a molecular weight of approximately 67-70 kDa and two light chains (L) having a molecular weight of approximately 25 kDa.

[0046] As used herein, "probe homogenizer" refers to a rotor / stator type homogenizer comprising a rotating blade member (rotor) and a fixed collar containing perforations (stator).

[0047] As used herein, “high-pressure homogenizer” refers to a homogenizing apparatus that includes a homogenization valve, which typically contains a narrow channel through which material is forced to flow, and a high-pressure pump, which pushes the material through the valve. High-pressure homogenizers typically operate at pressures of 5,000 psi to 45,000 psi.

[0048] Hyperimmunized egg products Oviparous animals produce antibodies in their blood and eggs that are specific to certain immunogens. For example, various genera of birds, such as chickens (Gallus domesticus), turkeys, and ducks, produce antibodies against antigens associated with avian diseases. LeBacq-Verheyden et al. (Immunology 27:683 (1974)) and Leslie, GA et al. (J. Med. 130:1337 (1969)) quantitatively analyzed chicken immunoglobulins. Polson, A. et al. (Immunological Communications 9:495-514 (1980)) immunized hens with several proteins and natural mixtures of proteins and detected IgY antibodies in the egg yolk. Fertel, R. et al. (Biochemical and Biophysical Research Communications 102:1028-1033 (1981)) immunized hens with prostaglandins and detected antibodies in egg yolk. Jensenius et al. (Journal of Immunological Methods 46:63-68 (1981)) provide a method for isolating egg yolk IgG for use in immunodiagnosis. Polson et al. (Immunological Communications 9:475-493 (1980)) describe antibodies isolated from egg yolk of hens immunized with various plant viruses.

[0049] U.S. Patent No. 4,748,018 (Stolle et al., 1988) discloses a method for passive immunization of mammals, comprising parenteral administration of purified antibodies obtained from the eggs of birds immunized against a corresponding antigen, wherein the mammals have acquired immunity to the eggs. U.S. Patent No. 5,772,999 (Greenblatt et al., 1998) discloses a method for preventing, counteracting, or mitigating chronic gastrointestinal disorders or nonsteroidal anti-inflammatory drug-induced (NSAID-induced) gastrointestinal injury in subjects by administration of highly immunized eggs and / or milk or fractions thereof.

[0050] Immunized eggs are eggs derived from birds that have been immunized, for example, with a specific antigen or a mixture of antigens. Highly immunized eggs are eggs derived from birds that have been brought to a specific immunized state, for example, by regular booster administration of antigens. Highly immunized eggs have been found to have various beneficial factors, as described above, including the treatment of chronic gastrointestinal disorders, NSAID-induced gastrointestinal injury (see U.S. Patent No. 5,772,999), and anti-inflammatory effects due to the presence of anti-inflammatory compositions (see U.S. Patent Application Publication No. US2004 / 0156857 (Adalsteinsson et al., 2004)).

[0051] One advantage of highly immunized egg products is that they will have higher and more consistent levels of antibodies (e.g., IgY antibodies) to one or more of the antigens described herein, compared to control egg products or egg products from chickens immunized with antigens using standard immunization techniques. Typically, standard immunization consists of an initial immunization, followed by one or two booster immunizations at 30-day intervals. In some embodiments, highly immunized egg products include at least four, five, six, seven, eight, nine, or ten immunizations with the antigens described herein. In some embodiments, highly immunized egg products include immunizing oviparous animals with the antigens described herein at intervals of less than 30 days, less than 25 days, less than 20 days, less than 15 days, less than 10 days, or less than 5 days. In some embodiments, highly immunized egg products include immunizing oviparous animals with the antigens described herein at intervals of one week, two weeks, three weeks, four weeks, one month, two months, or three months. Any of these values ​​can be used to define the range of intervals at which oviparous animals are immunized. For example, in some embodiments, ovipositors are highly immunized at intervals ranging from once every two weeks to once every three months, once a week to once every three months, or once every two weeks to once a month.

[0052] Highly immunized egg products may be produced by any egg-laying animal. The animal is preferably a member of the class Aves, or in other words, a bird. Among Aves, domesticated poultry is preferred, but other members of this class, such as turkeys, ducks, and geese, are suitable sources of highly immunized egg products. In certain embodiments, the egg-laying animal is a chicken.

[0053] This special state of high immunization is preferably achieved by administering primary immunization followed by periodic boosters using a sufficiently high dose of a specific antigen or mixture of antigens. The booster dose may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the dose required to induce primary immunization in ovipositors. Any of these percentages can be used to define a range for the booster immunization dose. For example, in some embodiments, the booster dose is 20% to 80%, 30% to 70%, or 50% to 100% of the dose required to induce primary immunization in ovipositors. In certain embodiments, the booster immunization dose is 50% of the primary immunization dose.

[0054] Given knowledge of the requirements for inducing and maintaining a highly immune state, it is within the scope of the skills in this art to vary the amount of antigen administered depending on the genus and strain of the ovipositor used to maintain a highly immune state in the animals.

[0055] A state of high immunity can be brought about by a single antigen or a combination of antigens. High immunization can be achieved by multiple exposures to multiple antigens or multiple exposures to a single antigen.

[0056] Advanced Immunization Procedures The following list of steps is an example of a preferred procedure used to immunize egg-laying animals so that the resulting highly immunized eggs or egg products can be administered to birds: 1. Select one or more antigens. 2. Primary immunization induces an immune response in egg-laying animals. 3. Administer an appropriate dose of one or more antigen booster vaccines to induce and maintain a high level of immunity.

[0057] Step 1: The key point in this step is that the antigen must be able to induce an immune state and a highly immune state in the ovipositor.

[0058] Step 2: The vaccine can be administered by any method that induces an immune response. Immunization is preferably achieved by administering the vaccine via intramuscular or subcutaneous injection. The preferred muscle for injection into birds is the pectoralis muscle. The dosage is preferably 0.05 to 5 milligrams of immunogenic vaccine. Other possible methods of administration include intravenous injection, intraperitoneal injection, intradermal, anal suppositories, aerosols, or oral administration.

[0059] Several methods known to those skilled in immunology can be used to determine whether a vaccine has induced an immune response in oviparous animals. Examples include enzyme-linked immunosorbent assay (ELISA), tests for the presence of antibodies against a stimulating antigen, and tests designed to assess the ability of a host to respond to an antigen. The minimum dose of antigen required to induce an immune response depends on the vaccination procedure used, including the type of adjuvant and antigen formulation used, as well as the type of oviparous animal used as the host.

[0060] Step 3: A highly immunized state is preferably induced and maintained in the target animal by repeated booster administration of appropriate doses at fixed time intervals. The time interval is preferably 1 week to 3 months over a period of 6 to 12 months. However, it is essential that the booster administration does not induce immune tolerance. Such processes are well known in the art. A method for preparing highly immunized egg products is described, for example, in U.S. Patent No. 6,803,035 (Greenblatt et al., 2004), which is incorporated herein by reference in its entirety.

[0061] In certain embodiments, the antigens described herein are formulated into vaccines containing an adjuvant. The adjuvant can be selected from Freund's complete adjuvant, Freund's incomplete adjuvant, saponins, biodegradable polymers, aluminum hydroxide, mineral oil, surfactants, and combinations thereof. Exemplary saponins include QS-21 and Quil A. Exemplary biodegradable polymers include chitosan, zymosan, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), e.g., Pluronic® L121 block copolymer, poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), polycaprolactone, and combinations thereof. Exemplary surfactants include polysorbate 80 and sorbitan trioleate. In some embodiments, the antigens described herein are formulated with an adjuvant selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, and QS-21 saponin. In the initial vaccination, oviparous animals receive two 0.5 mL doses of each antigen. Two weeks later, one 0.5 mL dose of each antigen is administered to the oviparous animals as a booster vaccination. An additional booster vaccination is administered four weeks after the initial vaccination. The vaccine can be administered intramuscularly. The vaccine can be administered into mammary tissue.

[0062] Other advanced immunization maintenance procedures or combinations of procedures, such as using intramuscular injection for primary immunization and intravenous injection for booster injection, are possible. Further procedures include the simultaneous administration of microencapsulated antigen and liquid antigen, or intramuscular injection for primary immunization and booster administration by oral or parenteral administration using microencapsulation means. Several combinations of primary and advanced immunization are known to those skilled in the art.

[0063] Highly immunized eggs or highly immunized egg products may contain increased levels of antibodies (e.g., IgY antibodies) specific to the particular antigens disclosed herein, compared to control eggs or control egg products obtained from ovipositors that have not been highly immunized with the particular antigen. In certain embodiments, the antibody is an IgY antibody.

[0064] In some embodiments, the highly immunized egg or egg product contains at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, or 500% more by weight of an antibody specific to a particular antigen (e.g., IgY antibody) as disclosed herein, compared to a control egg or control egg product obtained from an ovipositor that has not been highly immunized with the particular antigen.

[0065] Highly immunized eggs or highly immunized egg products may contain increased levels of antibodies against two or more of the antigens disclosed herein, compared to control eggs or control egg products obtained from non-immunized ovipositors.

[0066] The comparison of antibody titers in highly immunized egg products and control egg products can be determined by methods known in the art. For example, in one embodiment, eggs are collected and antibody titers are monitored at regular intervals by ELISA. To determine antibody titers, total IgY is extracted from eggs using the Pierce® Chicken IgY Purification Kit (Thermo Fisher Scientific, Waltham, WA). Briefly, 2 mL of egg is mixed with 5 times the volume of degreasing reagent, and IgY is purified according to the manufacturer's instructions. The spray-dried egg powder sample is reconstituted at 1 mg / mL in sterile PBS and filtered through a 0.22 μm membrane filter. The specific antibody titers in the isolated IgY or egg powder sample are measured by ELISA. A flat-bottomed 96-well microtiter plate (Corning® Costar®, Corning, NY) is coated with purified recombinant protein (e.g., antigens B, C, Co1, or Co2) at 10 μg / mL (100 μL / well) and incubated overnight at 4°C. The plate is washed twice with PBS containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO), blocked with 100 μL / well PBS containing 1% bovine serum albumin (BSA), and incubated at room temperature for 1 hour. IgY samples, serially diluted from egg powder samples (in PBS containing 0.1% BSA), are added to the plate in triple wells (100 μL / well) and incubated at room temperature for 2 hours with constant shaking. Next, the plates are washed with PBS-T and treated with peroxidase-conjugated rabbit anti-chicken IgY (IgG) antibody (1:500, Sigma), incubated for 30 minutes, and then colored for 10 minutes with 0.01% tetramethylbenzidine substrate (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0. The conjugated antibody is then measured at optical density (0D) at 450 nm using a microplate reader (Bio-Rad, Hercules, CA). 450 It is detected by measuring ).

[0067] Antibody titer can be expressed by the maximum dilution factor of the egg product that still contains detectable antibodies, as measured by optical density as described above. For example, an antibody titer of 1000 would indicate that a 1000-fold dilution of the egg product contains detectable antibodies, but higher dilutions do not. In some embodiments, the antibody titer in highly immunized egg products is at least 100,000, at least 250,000, at least 500,000, or at least 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 11 million, 12 million, 13 million, 14 million, 15 million, 16 million, 17 million, 18 million, 19 million, or 20 million.

[0068] In some embodiments, highly immunized eggs or egg products contain at least 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, or 0.1% by weight of IgY antibodies against a specific antigen. Typically, a whole chicken egg weighs about 60 grams excluding the shell, with the yolk weighing about 20 grams and the egg white weighing about 40 grams. In some embodiments, 3 grams of yolk contains about 25 milligrams of total IgY, so a whole egg contains about 150–200 mg of total IgY. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the total IgY in highly immunized eggs or egg products is specific to one of the antigens used for highly immunization.

[0069] Highly immunized eggs or egg products may contain increased levels of two or more antibodies (e.g., IgY antibodies) compared to control eggs or egg products obtained from non-immunized ovipositors, each of which is specific to a different antigen disclosed herein. The level of increase in each antibody (e.g., IgY antibody) in highly immunized eggs or egg products may be at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, or more by weight compared to control eggs or egg products.

[0070] Composition and administration Once ovipositors are sufficiently highly immunized, the eggs from these animals can be collected and processed to produce highly immunized egg products in an administerable form. Highly immunized egg products can be prepared by dehydration, spray drying, or freeze-drying of whole eggs, egg yolks, or purified IgY fractions. The dried highly immunized egg products can be mixed with pharmaceuticals, such as silicon or silicon derivatives that improve flow properties. The dried highly immunized egg products may contain a desiccant or be stored in a container containing a desiccant. Highly immunized egg products may be stored at ambient temperature or refrigerated, for example, at 4°C.

[0071] In some embodiments, highly immunized egg products can be encapsulated. In some embodiments, nanoparticles provided herein can be encapsulated. Methods for encapsulating antibodies and other proteins are known in the art, for example, described in U.S. Patent No. 7,105,158 (D'Souza et al., 2006). Biodegradable and non-antigenic materials can be used as encapsulation materials. Examples of encapsulation materials, but not limited to, include albumin, PLGA, globulin, natural and synthetic polymers, and thermoplastic polymers. Biocompatibility and biobiotechnology ErosiveAny polymer can be used for encapsulation. Several available crosslinking agents, such as glutaraldehyde, can be used to crosslink the encapsulation material. Furthermore, the nanospheres provided herein may be provided together with microspheres, the microspheres provided as two or more clusters, each cluster of microspheres having a different level of crosslinking, thereby resulting in the long-term continuous release of nanospheres containing polypeptides such as IgY.

[0072] Microparticle and nanoparticle formulations In certain embodiments, the Disclosure relates to nanoparticles comprising a composition comprising a polypeptide, wherein the composition is biocompatible with bio-particles. erosiveness This invention relates to nanoparticles encapsulated in polymers and surfactant polymers, such as PVA, polyvinylpyrrolidone, or combinations thereof. Nanoparticles are colloidal particles with a size range of nanometers. Due to their smaller size, they have a larger surface area, which tends to result in higher loading efficiency and further enhances the bioavailability of polypeptides contained within the nanoparticles.

[0073] In some embodiments, the polypeptide is a therapeutic polypeptide, i.e., a polypeptide administered to a subject for the treatment of a disorder. In some embodiments, the polypeptide is an antibody, such as a polyclonal antibody or a monoclonal antibody. In some embodiments, the antibody is IgY. In some embodiments, IgY is obtained from highly immunized eggs. In some embodiments, the composition encapsulated in nanoparticles is a highly immunized egg product containing IgY. In some embodiments, the composition contained within the nanoparticles is a defatted fraction from egg yolk containing at least 95 wt%, 96 wt%, 97 wt%, 98 wt%, or 99 wt% IgY based on the total weight of the defatted egg yolk fraction. In some embodiments, the composition contained within the nanoparticles contains purified IgY.

[0074] Nanoparticles can be prepared using one or more polymers. Suitable polymers include, but are not limited to, polyacrylates, polymethacrylates, polycarbonates, polypropylenes, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl halides, polysiloxanes, polyurethanes and their copolymers, hydroxyalkylcellulose, cellulose ethers, nitrocellulose, methylcellulose, ethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate / butyrate, cellulose triacetate, sodium cellulose sulfate, and others. Examples include poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, poly(ethylene terephthalate), poly(vinyl acetate), and polyvinyl chloride, polystyrene, as well as mixtures, copolymers, and blends thereof. In certain embodiments, the polymer includes polymethacrylate.

[0075] In some embodiments, the polymer used to form nanoparticles is EUDRAGIT® L 100 (methacrylic acid copolymer L (methacrylic acid-methyl methacrylate copolymer (1:1), or methacrylic acid copolymer, type A, available from Evonik Corporation, Allentown, PA). The ratio of free carboxyl groups to ester groups is approximately 1:1 in EUDRAGIT® L 100.

[0076] In some embodiments, the polymer used to form nanoparticles or microparticles is EUDRAGIT® S 100 (methacrylic acid copolymer S (methacrylic acid-methyl methacrylate copolymer (1:2), or methacrylic acid copolymer, type B, available from Evonik Corporation, Allentown, PA). The ratio of free carboxyl groups to ester groups is approximately 1:2 in EUDRAGIT® S 100.

[0077] In some embodiments, a combination of EUDRAGIT® L 100 and EUDRAGIT® S 100 can be used to form nanoparticles or microparticles. The ratio of EUDRAGIT® L 100 to EUDRAGIT® S 100 may be in the range of 99:1 to 1:99. In some embodiments, the ratio of EUDRAGIT® L 100 to EUDRAGIT® S 100 may be 80:20 to 20:80, or 75:25 to 25:75, or 60:40 to 40:60. In some embodiments, EUDRAGIT® L 100 is used in greater amounts than EUDRAGIT® S 100 (there is a greater wt% of EUDRAGIT® L 100 than EUDRAGIT® S 100). In some embodiments, the ratio of EUDRAGIT® L 100 to EUDRAGIT® S 100 is in the range of 2:1 to 10:1. In some embodiments, more EUDRAGIT® S 100 is used than EUDRAGIT® L 100 (there is a greater wt% of EUDRAGIT® S 100 than EUDRAGIT® L 100). In some embodiments, the ratio of EUDRAGIT® S 100 to EUDRAGIT® L 100 is in the range of 2:1 to 10:1. In some embodiments, equal amounts of EUDRAGIT® L 100 and EUDRAGIT® S 100 are used.

[0078] The pH-responsive release profile for nanoparticles is related to the biocompatible bio-based materials used to form the nanoparticles. erosiveness This can be achieved by the appropriate selection of one or a combination of polymers as the polymer.

[0079] When forming nanoparticles or microparticles, surfactant polymers may also be included. Exemplary surfactant polymers include poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof. In some embodiments, the surfactant polymer includes PVA.

[0080] By encapsulating IgY within nanoparticles, IgY becomes more stable and can retain its natural structure without degradation. The use of nanoparticles ensures that IgY trapped within the nanoparticles is safe not only in gastric juice but also in intestinal juice, and is protected from enzymatic degradation. Because it is trapped within nanoparticles, the molecular weight and large size of IgY are no longer a problem, given that the overall size of the nanoparticles is in the nanoscale range. Therefore, it can be more easily presented to and passed through cells or interstitial regions to reach target sites.

[0081] Nanoparticles may have any shape. Typically, nanoparticles are spherical. Other suitable shapes include, but are not limited to, flake, triangular, elliptical, rod-shaped, polygonal, needle-shaped, tubular, cubic, and rectangular parallelepiped structures. In certain embodiments, nanoparticles have a diameter of less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, or 10 nm. Any of these values ​​can be used to define a range of nanoparticle diameters. For example, the diameter of nanoparticles may be about 10 to about 1000 nm, about 100 to about 1000 nm, or about 50 to about 500 nm, or about 10 to about 100 nm, about 200 to about 300 nm, about 250 to about 300 nm, or about 100 to about 400 nm. In certain embodiments, nanoparticles have a diameter of less than 300 nm.

[0082] The concentration of the polypeptide (e.g., IgY) encapsulated in the nanoparticles can be expressed as a loading percentage, i.e., the ratio of the weight of the polypeptide (e.g., IgY) to the weight of the nanoparticles multiplied by 100. In some embodiments, the nanoparticles contain about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 wt% of the polypeptide (e.g., IgY) relative to the total weight of the nanoparticles. In some embodiments, the nanoparticles contain less than approximately 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 wt% of polypeptides (e.g., IgY) relative to the total weight of the nanoparticles. In some embodiments, the nanoparticles contain at least about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 wt% of polypeptide (e.g., IgY) relative to the total weight of the nanoparticles. Any of these values ​​can be used to define a range of polypeptide (e.g., IgY) concentrations in the nanoparticles. For example, the nanoparticles may contain polypeptide (e.g., IgY) at concentrations ranging from about 1 wt% to about 10 wt%, or from about 1 wt% to about 5 wt%. In some embodiments, the concentration of polypeptide (e.g., IgY) in the nanoparticles is at least about 5 wt%.

[0083] In some embodiments, nanoparticles containing polypeptides (e.g., IgY) are anionic and, for example, have a negative zeta potential. In some embodiments, the zeta potential of the nanoparticles is less than 0, -1, -5, -10, -15, -20, -25, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -45, or -50 mV. Any of these values ​​can be used to define a range of zeta potentials for the nanoparticles. For example, in some embodiments, the zeta potential of the nanoparticles is about 0 to about -40 mV, about 0 to about -50 mV, about -10 to about -50 mV, about -20 to about -50 mV, or about -30 to about -40 mV. In some embodiments, the zeta potential of the nanoparticles is about -38.9 mV. In some embodiments, the zeta potential of the nanoparticles is about -31.2 mV.

[0084] For example, several processes exist for preparing nanoparticles, including multilayer microencapsulation, hot-melt encapsulation, phase-separation encapsulation, spontaneous emulsification, solvent evaporation microencapsulation, solvent removal microencapsulation, and coacervation. These methods are known in the art. A detailed description of these methods is discussed in Mathiowitz et al., “Microencapsulation”, in Encyclopedia of Controlled Drug Delivery, vol. 2, pp. 495-546, 1999, John Wiley & Sons, Inc., New York, NY, which is incorporated herein by reference in its entirety.

[0085] A method is provided for preparing nanoparticles containing polypeptides having a molecular weight greater than 50,000 g / mol. For example, in some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticles is at least 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, or 200,000 g / mol. In some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticles is 50,000 to 100,000 g / mol, 50,000 to 180,000 g / mol, or 50,000 to 200,000 g / mol. In some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticles is approximately 180,000 g / mol.

[0086] A combination of homogenization processes is used to prepare nanoparticles. A probe homogenizer can be used to prepare the initial emulsion, which can then be processed in a high-pressure homogenizer.

[0087] A probe homogenizer is a rotor-stator homogenizer comprising a rotating blade member (rotor) and a fixed collar containing perforations (stator). The rotating blade pushes the material through the perforations, and as the material passes through the perforations, the shear force between the perforations in the stator and the rotating blade reduces the particle size. The movement of the blades also draws the material into the rotor / stator assembly, which also causes cavitation, which can also help reduce the particle size. The size of the perforations in the stator, the rotational speed of the rotating blade member, the viscosity of the material, and the length of processing time can affect the resulting particle size. Probe homogenizers with various dimensions and configurations of rotors and stators are known and commercially available in the art and can be used in the methods provided herein.

[0088] Next, the emulsion produced using a probe homogenizer can be processed in a high-pressure homogenizer (HPH). A high-pressure homogenizer converts flow pressure into kinetic energy. A typical high-pressure homogenizer includes a homogenization valve that forces the material through it, and a high-pressure pump that pushes the material through the valve. The high-pressure homogenization process exposes the material being processed to several forces, including shear, cavitation, turbulence, impact force, and high pressure, which helps in the formation of nanoparticles, potentially resulting in nanoparticles with optimal characteristics such as minimum particle size, good particle size distribution, and maximum polypeptide loading. High-pressure homogenizers of various dimensions and configurations are known and commercially available in the art and can be used in the methods provided herein.

[0089] In exemplary embodiments, the aqueous phase may be prepared to contain a composition comprising a polypeptide (e.g., IgY) and a surfactant polymer. The surfactant polymer may be poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), or a combination thereof. The organic phase is then prepared. The organic phase may include anhydrous ethanol, dichloromethane (DCM), acetonitrile, acetone, ethyl acetate, chloroform, and combinations thereof. In some methods, the organic phase includes DCM. erosiveness A solution or suspension containing a polymer is prepared. To aid mixing, an aqueous phase or a biocompatible bio-phase is added. erosiveness A polymer solution or suspension, or both, may be subjected to sonication. Then, an aqueous phase, an organic phase, and a biocompatible biological... erosivenessA polymer solution or suspension may be mixed together and subjected to emulsification using a probe homogenizer (rotor / stator homogenizer). The composition and duration of the mixing may depend on the volume of the material being processed. In some methods, the mixture is subjected to homogenization using a probe homogenizer at a rotor rotation speed of 5,000 to 15,000 rpm. The processing time may be 1 to 10 minutes. Cooling elements may be included during probe homogenization. For example, to remove heat from the material being processed during probe homogenization, the container containing the material being processed may be placed in an ice bath, or a cooling system may be used. Processing with a probe homogenizer yields a crude emulsion.

[0090] The crude emulsion is then subjected to a further homogenization process using a high-pressure homogenizer. The pressure used may be approximately 5,000 psi to 45,000 psi. In some embodiments, the method includes processing in a high-pressure homogenizer at a pressure of approximately 15,000 psi.

[0091] Next, the emulsion obtained from the high-pressure homogenizer can be subjected to solvent removal. In some embodiments, a rotary evaporator can be used to remove the solvent. Any rotary evaporator known in the art can be used to remove the solvent. In some embodiments, a rotary vacuum evaporator can be used. The evaporation time of the solvent may depend on the amount of solvent present and the chemical properties of the solvent. In some embodiments, only the organic solvent is removed, leaving an aqueous suspension of nanoparticles. In some methods, rotary vacuum evaporation at a pressure of about 50 to 250 psi is used. In some methods, rotary vacuum evaporation at a pressure of about 200 psi for about 10 minutes is used to remove the organic solvent. In some methods, the emulsion from the high-pressure homogenizer may become clear after rotary vacuum evaporation, indicating that the organic solvent has been removed.

[0092] The material obtained after removal of the organic solvent can be subjected to centrifugation to recover the nanoparticles. In some methods, a two-step centrifugation process is available. In a typical two-step centrifugation process, the obtained material can first be centrifuged at 3,000–5,000 × g for 2–10 minutes. In some embodiments, the material obtained after solvent removal can be centrifuged at 4,400 × g for about 3 minutes. After the first centrifugation step, the supernatant can be collected and centrifuged at 20,000–25,000 × g for 10–30 minutes using a high-speed centrifuge such as an Avanti® JE high-speed centrifuge. In some methods, after the first centrifugation step, the supernatant can be collected and centrifuged at about 22,000 × g for about 15 minutes using a high-speed centrifuge. The pelletized nanoparticles can be resuspended in Milli-Q® IQ water (produced using a Milli-Q® IQ water purification system available from MilliporeSigma, St. Louis, MO).

[0093] In some embodiments, a concentrated polypeptide (e.g., IgY) is encapsulated within nanoparticles. For example, in some embodiments, the polypeptide (e.g., IgY) is purified or partially purified and concentrated before the formation of nanoparticles. Methods for purifying and concentrating IgY antibodies from egg products are known in the Art, for example, described in U.S. Patent No. 5,367,054 (Lee, 1994), which is incorporated herein by reference in whole.

[0094] In certain embodiments, nanoparticle compositions containing the nanoparticles disclosed herein may be administered to a subject orally. Eggs and egg yolks are natural food materials, non-toxic, and safe for oral ingestion. Dosage forms containing or containing nanoparticles provided herein may further include a pharmaceutically acceptable carrier suitable for oral administration. In some embodiments, the pharmaceutically acceptable carrier includes compounds generally recognized as safe by the FDA (GRAS). In some embodiments, the GRAS compounds include acetic acid, aconitic acid, adipic acid, alginic acid, and Bacillus stearothermophilus. α-amylase enzyme preparation derived from Stearothermophilus, benzoic acid, bromelain, caprylic acid, mixed product of carbohydrase and protease enzymes, citric acid, catalase (bovine liver), lactic acid, enzyme-modified lecithin, linoleic acid, malic acid, potassium bitartrate, propionic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, diacetyl tartrate esters of monoglycerides and diglycerides, agar, brown algae, red algae, ammonium alginate, ammonium bicarbonate, ammonium carbonate, ammonium chloride, ammonium hydroxide, dibasic ammonium citrate, monobasic ammonium phosphate, ri Ammonium ammonium sulfate (dibasic), ammonium sulfate, bacterial carbohydrase enzyme preparation, bacterial protease enzyme preparation, bentonite, benzoyl peroxide, n-butane and isobutane, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium glycerophosphate, calcium hydroxide, calcium iodate, calcium lactate, calcium oxide, calcium pantothenate, calcium propionate, calcium stearate, calcium sulfate, carbon dioxide, beta-carotene, Trichoderma longibrachiatumCellulase enzyme preparation derived from *Longibrachiatum*, clove and its derivatives, cocoa butter substitute, copper gluconate, copper sulfate, corn silk and corn silk extract, cuprous iodide, L-cysteine, L-cysteine ​​monohydrochloride, dextrin, diacetyl, dill and its derivatives, enzyme-modified fat, ethyl alcohol, ethyl formate, ferric ammonium citrate, ferric chloride, ferric citrate, ferric phosphate, ferric pyrophosphate, ferric sulfate, ferrous ascorbate, ferrous carbonate, ferrous citrate, ferrous fumarate, ferrous gluconate, ferrous lactate, sulfuric acid Ferrous iron, ficin, garlic and its derivatives, glucono delta-lactone, corn gluten, wheat gluten, glyceryl monooleate, glyceryl monostearate, glyceryl behenate, glyceryl palmitostearate, acacia (gum arabic), gatchi gum, guar gum, locust (carob) bean gum, karaya gum (sterclear gum), tragacanth gum, gellan gum, xanthan gum, hydrogen peroxide, inositol, insoluble glucose isomerase enzyme preparation, elemental iron, isopropyl citrate, lactase enzyme preparation derived from Candida pseudotropicalis, lactase enzyme preparation derived from Kluyveromyces lactis, lecithin, licorice and licorice derivatives, crushed limestone, animal lipase, Rhizopus niveusLipase enzyme preparation derived from niveus, magnesium carbonate, magnesium chloride, magnesium hydroxide, magnesium oxide, magnesium phosphate, magnesium stearate, magnesium sulfate, malt, maltodextrin, malt syrup (malt extract), manganese chloride, manganese citrate, manganese gluconate, manganese sulfate, menhaden oil, methylparaben, microparticle protein products, monoglycerides and diglycerides, monosodium phosphate derivatives of monoglycerides and diglycerides, niacin, niacinamide, nickel, niacin preparations, nitrogen, nitrous oxide, peptone, rapeseed oil, bovine bile extract, ozone, pancreatin, papain, pectin, pepsin, potassium alginate, potassium bicarbonate, potassium carbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium iodide, potassium iodate, potassium lactate, potassium sulfate, propane, propyl gallate, propylene glycol, propylparaben, propyl Lidoxine hydrochloride, rennet (animal-derived) and chymosin preparation (fermentation-derived), riboflavin, riboflavin-5-phosphate (sodium), rue, rue oil, shea nut oil, sodium acetate, sodium alginate, sodium benzoate, sodium bicarbonate, sodium carbonate, sodium citrate, sodium diacetate, sodium hydroxide, sodium hypophosphate, sodium lactate, sodium metasilicate, sodium propionate, sodium sesquicarbonate, sodium tartrate, sodium potassium tartrate, sodium thiosulfate, sorbitol, stannous chloride (anhydrous and dihydrate), starter distillate, stearyl citrate, sucrose, corn sugar, invert sugar, corn syrup, high fructose corn syrup, thiamine hydrochloride, thiamine mononitrate, α-tocopherol, triacetin, triptyline, triethyl citrate, trypsin, urea, Lactobacillus fermentum Urease enzyme preparation derived from Lactococcus fermentum, vitamin A, vitamin B12, vitamin D, beeswax (yellow and white), candelilla wax, carnauba wax, whey, low-lactose whey, low-mineral whey, whey protein concentrate, baker's yeast extract, brewer's yeast extract, zein, and Lactococcus lactis.The aminopeptidase enzyme preparations derived from lactis, or any combination thereof, are selected.

[0095] In certain embodiments, a dosage form containing nanoparticles or a composition containing nanoparticles provided herein may contain one or more additional compounds, such as nutrients or probiotics. For example, in one embodiment, nanoparticles or a composition containing nanoparticles can be incorporated into a dietary supplement.

[0096] One method for preparing highly immunized eggs to be incorporated into nanoparticles involves drying the eggs to egg powder. Various methods for drying eggs are known, including freeze-drying and spray-drying. In one method, spray-drying is used. The process of spray-drying eggs is well known in the art.

[0097] In certain embodiments, the whole egg is separated into separate fractions, such as yolk and egg white. For example, it is generally known in the art that IgY antibodies are found in the yolk. Therefore, those skilled in the art will clearly recognize that separation of the yolk can provide a more effective fraction or remove undesirable components. Such further separation provides the ability to produce nanoparticles containing a more effective egg fraction. In some embodiments, IgY is separated from a highly immunized egg product and incorporated into the nanoparticles. The separated IgY can be processed into a freeze-dried or spray-dried powder to be incorporated into the nanoparticles. The IgY can be further purified before being incorporated into the nanoparticles.

[0098] In certain embodiments, the Disclosure relates to a method for delivering a polypeptide (e.g., IgY) to a subject, comprising the step of administering nanoparticles or compositions containing nanoparticles as described herein to the subject by oral administration. The nanoparticles or compositions containing nanoparticles are preferably administered to the subject in an amount effective to treat or prevent a particular disorder. The dosage and duration of administration depend on the particular condition of the subject. In some embodiments, the nanoparticles or compositions containing nanoparticles are administered to the subject for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 60, 90, 180, or 365 days. The nanoparticles or compositions containing nanoparticles may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more per day. Any of these values ​​can be used to define the range of times the composition may be administered to the subject per day. For example, in some embodiments, nanoparticles or compositions containing nanoparticles are administered to the subject 1 to 2 times per day, 1 to 3 times per day, or 1 to 4 times per day. In some embodiments, nanoparticles or compositions containing nanoparticles are administered to the subject at least 2 times per day. In some embodiments, nanoparticles or compositions containing nanoparticles are administered to the subject at least 1 time per day.

[0099] In some embodiments, the composition is administered to the subject daily. In some embodiments, the nanoparticles or the composition containing nanoparticles is administered to the subject every two days. In some embodiments, the nanoparticles or the composition containing nanoparticles is administered to the subject every three days. In some embodiments, the nanoparticles or the composition containing nanoparticles is administered to the subject once a week. In certain embodiments, the nanoparticles or the composition containing nanoparticles is administered to the subject once a day for more than 10 consecutive days. The use of the nanoparticles or the compositions containing nanoparticles described herein for delivering polypeptides to a subject is also provided.

[0100] In some embodiments, a daily dose (or a highly immunized egg product containing an equivalent amount of one to a few highly immunized whole eggs) may be administered to the subject depending on the specific circumstances of their condition.

[0101] In certain embodiments, the effective dose of the highly immunized egg product in the form of nanoparticles administered to a subject (e.g., a human) is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams per day. For example, in some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams of whole egg nanoparticles are administered to the subject per day. In some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams of egg yolk nanoparticles are administered to the subject per day. In some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 grams of dried egg yolk or dried whole egg nanoparticles are administered to the subject per day. Any of these values ​​can be used to define the range of effective doses of nanoparticles containing highly immunized egg products administered to mammals. For example, in some embodiments, the effective dose of nanoparticles containing highly immunized egg products is between 0.1 and 10 grams, between 0.5 and 6 grams, or between 1 and 5 grams per day. In certain embodiments, an amount of nanoparticles containing IgY equivalent to the amount of IgY in 3 grams of egg yolk is administered to a subject (e.g., a human) per day. [Examples]

[0102] Example 1. IgY quantification using NanoDrop(trademark)One IgY and serial dilutions of IgY ranging from 0.105 to 26.9 mg / mL were analyzed for A280 (absorbance at 280 nm) values ​​using a NanoDrop® One Microvolume UV-Vis spectrophotometer (Thermo Scientific). IgY was selected as the sample type, and baseline correction was performed at 320 nm. 2 μL of 1× phosphate-buffered saline (PBS) was placed on the lower base and lower arm for blanking (automatic blanking was on). Samples were gently vortexed before measurement. Each 2 μL aliquot of the sample was loaded, and the respective A280 value was recorded. All sample dilutions were performed using 1× PBS. The A280 values ​​are shown in Table 1.

[0103] [Table 1]

[0104] The results are shown graphically in Figure 1, which provides a calibration curve for IgY quantification. 2 The value was determined to be 0.9999.

[0105] Example 2. Preparation of IgY nanoparticles Nanoparticles containing IgY antibody were prepared. A high-pressure homogenizer (HPH) was used to achieve efficient loading of IgY into the nanoparticles. The high-pressure homogenization process facilitates exposure of the formulation to multiple forces, including shear, cavitation, turbulence, impact force, and high pressure, which helps achieve nanoparticles with optimal characteristics such as minimum particle size and maximum drug loading. EUDRAGIT® L 100 was used either alone or in combination with EUDRAGIT® S 100 to prepare the IgY nanoparticles. An aqueous phase containing an IgY solution equivalent to 20 mg of IgY and 1% polyvinyl alcohol (PVA) was prepared. PVA was used as a surfactant polymer. An organic phase containing 3 mL of dichloromethane (DCM) and 1 mL of anhydrous ethanol was prepared. The EUDRAGIT® polymer was dissolved in anhydrous ethanol using an ultrasonic bath. Solutions containing IgY and PVA, a solution containing DCM, and an alcohol solution of EUDRAGIT® polymer were subjected to probe homogenization at 10,000 rpm for 1 minute using a digital high-speed homogenizer (Fisherbrand® 850 Homogenizer, Fisher Scientific, Waltham, MA). The resulting crude emulsions were processed through HPH (NanoDeBee homogenizer, BEE International, San Diego, CA). The detailed HPH parameters used for homogenization were as follows: nozzle type: N5; pressure: 15,000 psi; flow pattern: reverse; number of reactors: 6; number of cycles: 7. During high-pressure homogenization, Milli-Q® water (produced using a Milli-Q® IQ water purification system available from MilliporeSigma, St. Louis, MO) was added, and a 45 mL preparation was collected. Next, the formulation was subjected to rotary vacuum evaporation at approximately 200 psi for approximately 10 minutes to remove the organic solvent. The endpoint was determined when the solution became clear. The resulting solution was centrifuged at 4,400 × g for 3 minutes.The supernatant was collected and centrifuged at 22,000 × g for 15 minutes using an Avanti® JE high-speed centrifuge (Beckman Coulter Life Sciences, Indianapolis, IN). The collected nanoparticles were resuspended in 1 mL of Milli-Q® water.

[0106] Example 3. Physicochemical characterization of IgY nanoparticles IgY nanoparticles were analyzed for their physicochemical characteristics. The IgY nanoparticles were frozen overnight at -80°C. The frozen IgY nanoparticles were then subjected to lyophilization for 24 hours using an L Benchtop Freeze Dry system. The lyophilized IgY nanoparticle powder was collected, a known amount was dissolved in pH 7.4 buffer, and then the IgY content was analyzed by NanoDrop®One analysis.

[0107] A 10 μL nanoparticle formulation was diluted with 2 mL of Milli-Q® water and sonicated for 30 minutes. Particle size, polydispersity index (PDI), and zeta potential were measured using a Malvern zetasizing system. The capture efficiency (%EE) of IgY-loaded nanoparticles was also determined.

[0108] IgY nanoparticles formulated with EUDRAGIT® L 100 (IgY-L nanoparticles) were found to have an average particle size of 276.0 nm and a polydispersity index (PDI) of 0.22. IgY nanoparticles formulated with EUDRAGIT® L 100 / S 100 (IgY-L:S nanoparticles, with a L100:S100 ratio of 4:1) were found to have an average particle size of 272.1 nm and a polydispersity index (PDI) of 0.17. These data suggest that IgY nanoparticles have a uniform particle size distribution.

[0109] The zeta potential of IgY-L nanoparticles was found to be -38.9 mV, and the zeta potential of IgY-L:S nanoparticles was found to be -31.2 mV. Therefore, the nanoparticles were anionic. Anionic nanoparticles have higher stability during storage and higher safety during administration to humans or animals, as cationic nanoparticles can cause toxicity and cell death.

[0110] IgY was effectively encapsulated in nanoparticles, achieving encapsulation efficiencies (EE) of 35.03 ± 3.8% for IgY-L nanoparticles and 36.4 ± 1.99% for IgY-L:S nanoparticles. Encapsulation efficiency is the percentage of IgY in the sample encapsulated within the nanoparticles.

[0111] Example 4. ELISA assay Functional ELISA assays were performed using IgY extracted from nanoparticles and compared with control IgY at various concentrations. IgY-loaded nanoparticle formulations (IgY-L and IgY-L:S nanoparticles) were dissolved, and the dissolved samples were diluted to obtain a concentration of 300 ng / mL for IgY. The diluted samples were then quantified for the amount of IgY using an ELISA assay. Briefly, the antigen was diluted to 1 mg / mL in bicarbonate buffer pH 9.0. 100 μL of this antigen solution was added to the ELISA plate wells in a 96-well plate. The plate was then covered with Parafilm and incubated overnight at 4°C. The solution was then discarded by tilting / flicking the plate, followed by a single wash with 200 μL of PBS (PBST) containing 0.05 wt% Tween-20. Then, 200 μL of fresh blocking reagent (1% BSA in 1× PBS) was added to each well and incubated at 37°C for 1 hour. Next, the plate was washed once with PBST for 5 minutes. Then, it was tilted and gently tapped on a paper towel. Primary antibody from the diluted stock solution was added in PBST. Serial dilutions of IgY were performed in the range of 37–300 ng / mL, and the plate was incubated at 37°C for 1 hour. The plate was then washed three times with PBST, each wash being performed for 5 minutes using 100 μL of PBST without agitation. Diluted secondary antibody was added in blocking solution and incubated at 37°C for 1 hour, followed by two washes with PBST, and finally one wash with PBS alone, each wash for 5 minutes. Then, 100 μL of chromogenic solution (TMB substrate solution) was added and incubated at room temperature for 15–20 minutes, followed by the addition of 100 μL of stop solution. The absorbance of the plate was read at 450 nm using a plate reader.

[0112] A calibration was constructed on the plot for the quantification of IgY and its serial dilutions using a functional ELISA assay. The resulting equation was y = 0.0058x + 1.8906. The amount of IgY was calculated by incorporating the absorbance values ​​obtained from the soluble samples of IgY nanoparticles into the above equation. From the results, it was determined that IgY-L nanoparticles contained 274.2 ± 6.7 ng / mL of IgY antibody, and IgY-L:S nanoparticles contained 273.8 ± 6.4 ng / mL of IgY antibody. Regarding the loading percentage of the nanoparticles, IgY-L nanoparticles contained 5.84% (w / w) ± 0.64 IgY per nanoparticle weight, and IgY-L:S nanoparticles contained 6.07% (w / w) ± 0.33 IgY per nanoparticle weight.

[0113] Example 5. Preparation of capsules loaded with IgY nanoparticles Lipod-dried IgY nanoparticles were manually filled into size #3 rigid gelatin capsules. The loaded capsules were coated with either coating formulation A or B. The composition of coating formulation A is shown in Table 2. The composition of coating formulation B is shown in Table 3.

[0114] [Table 2]

[0115] [Table 3]

[0116] The actual amounts of ingredients for formulation can be appropriately scaled up according to the batch size to be manufactured. The required amounts of EUDRAGIT® L100 (0.6 g) [Formulation A] or EUDRAGIT® L100 (0.048 g), EUDRAGIT® S 100 (0.012 g) [Formulation B], talc (0.3 g), and triethyl citrate (0.06 g) were weighed. The required amounts of solvent: isopropyl alcohol (5.1 mL), milli-Q water (0.42 mL), and acetone (3.4 mL) were weighed. All weighed ingredients were added to the mixture of solvents and subjected to an ultrasonic treatment bath for 30 minutes. The resulting coating composition was transferred to a 25 mL glass beaker. Capsules containing IgY nanoparticles were then immersed in the coating formulation. The immersion coating was performed three times with a 5-minute drying step between coatings. Next, the capsules were dried using an air dryer with a hot airflow. Then, the integrity of the coated capsules was checked, and it was determined that the coating process did not affect the integrity of the capsules.

[0117] Example 6. In vitro release study Release studies of coated blank capsules and coated capsules containing IgY (Formulation A: EUDRAGIT® L100, and Formulation B: EUDRAGIT® L 100 and S 100) were performed in pH 1.2 and pH 7.0 buffers (50 mL volume) at 37°C. Coated capsules loaded with IgY nanoparticles were placed in the release medium (50 mL) using a sinker, and 1 mL of sample was collected at predetermined time points (pH 1.2: 1 and 2 hours, pH 7.0: 1 hour). After 2 hours at pH 1.2, the buffer was removed and replaced with pH 7.0 buffer. The collected samples were centrifuged at 30,000 rpm for 15 minutes and then filtered using a 0.2 μm syringe filter. The filtrate was analyzed using a NanoDrop® One spectrophotometer to determine the amount of IgY released from the capsules.

[0118] Capsules containing IgY nanoparticles and coated with coating formulation A were found to release approximately 46.3% of IgY after 1 hour and 99.0% after 2 hours in a pH 1.2 buffer. After replacing the pH 1.2 buffer with a pH 7.0 buffer, approximately 6.5% of IgY was released after 1 hour. Capsules containing IgY nanoparticles and coated with coating formulation B were found to release approximately 90.7% of IgY after 1 hour and 91.3% after 2 hours in a pH 1.2 buffer. After replacing the pH 1.2 buffer with a pH 7.0 buffer, approximately 11.5% of IgY was released after 1 hour. The following are examples of the forms of this disclosure: [1] Nanoparticles comprising a composition comprising a polypeptide, wherein the composition is encapsulated in a material comprising a biocompatible bioerosive polymer and a surfactant polymer. [2] The nanoparticle according to embodiment 1, wherein the biocompatible bio-erosive polymer is polymethacrylate. [3] The nanoparticles according to embodiment 1, wherein the nanoparticles have a diameter of less than 300 nm. [4] The nanoparticle according to embodiment 1, wherein the nanoparticle contains at least 5% w / w of polypeptide. [5] Nanoparticles according to any one of embodiments 1 to 4, wherein the polypeptide is IgY. [6] The nanoparticles according to embodiment 5, wherein IgY is obtained from highly immunized eggs. [7] Nanoparticles according to any one of embodiments 1 to 4, wherein the composition is a highly immunized egg product. [8] Nanoparticles according to any one of embodiments 1 to 4, wherein the composition is a defatted fraction from egg yolk containing at least 95% by weight of IgY. [9] Nanoparticles according to any one of embodiments 1 to 4, wherein the composition comprises purified IgY.

[10] Biocompatible bio-erosive polymers, (a) Methacrylic acid-methyl methacrylate copolymer (1:1), or (b) Methacrylic acid-methyl methacrylate copolymer (1:2), or (c) Both (a) and (b) A nanoparticle according to any one of embodiments 1 to 9, including the following:

[11] Nanoparticles according to any one of embodiments 1 to 10, wherein the surfactant polymer is selected from chitosan, poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

[12] Nanoparticles according to any one of embodiments 1 to 11, and Pharmacologically acceptable carriers A pharmaceutical composition containing the above.

[13] The pharmaceutical composition according to embodiment 12, wherein the pharmaceutically acceptable carrier is suitable for oral administration.

[14] The pharmaceutical composition according to embodiment 12 or 13, wherein the pharmaceutical composition is in a form selected from the group consisting of powder, tablets, enteric coated tablets, capsules, enteric coated capsules, suspensions, solutions, and oral beverages.

[15] A method for delivering a polypeptide, A step of administering nanoparticles described in any one of embodiments 1 to 11 to a target by oral administration, or A step of administering a pharmaceutical composition according to any one of embodiments 12 to 14 to a subject by oral administration. Methods that include...

[16] The method according to embodiment 15, wherein the polypeptide contained in the nanoparticles is IgY.

[17] The method according to embodiment 16, wherein IgY is obtained from highly immunized eggs.

[18] The method according to embodiment 15, wherein the composition contained in the nanoparticles is a highly immunized egg product.

[19] The method according to embodiment 15, wherein the composition contained in the nanoparticles is a defatted fraction from egg yolk containing at least 95% by weight of IgY.

[20] The method according to embodiment 15, wherein the composition contained in the nanoparticles contains purified IgY.

[21] The method according to any one of embodiments 15 to 20, wherein the biocompatible bio-erosive polymer of nanoparticles comprises polymethacrylate.

[22] The method according to any one of embodiments 15 to 21, wherein the nanoparticles have a diameter of less than 300 nm.

[23] The method according to any one of embodiments 15 to 22, wherein the nanoparticles comprise at least 5% w / w of polypeptide.

[24] (a) A step of preparing a first mixture comprising a polypeptide composition, water, and a surfactant polymer, (b) The step of ultrasonically treating the first mixture, (c) Adding a biocompatible bio-erosive polymer to ethanol and sonicating it to produce a second mixture, (d) Mixing the first mixture and the second mixture with an organic solvent to form a third mixture, (e) Homogenize the third mixture using a probe homogenizer at a speed of approximately 5,000 to 15,000 rpm to produce a crude emulsion. (f) The crude emulsion is subjected to high-pressure homogenization using a high-pressure homogenizer at a pressure of approximately 10,000 psi to 30,000 psi to form a fourth mixture, and (g) Evaporate the ethanol and organic solvent from the fourth mixture to produce a suspension containing nanoparticles. A method for preparing nanoparticles according to embodiment 1, including the following.

[25] The method according to embodiment 24, further comprising the step of centrifugating the suspension to obtain nanoparticles.

[26] The method according to embodiment 24 or 25, wherein the surfactant polymer is selected from chitosan, poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

[27] The method according to any one of embodiments 24 to 26, wherein the organic solvent comprises dichloromethane.

[28] The method according to any one of embodiments 24 to 27, wherein the first mixture comprises about 2% to 5% (w / w) of polypeptide.

[29] The method according to any one of embodiments 24 to 28, wherein the polypeptide is IgY.

[30] The method according to any one of embodiments 24 to 29, wherein the second mixture comprises 10 mg / mL to 20 mg / mL of a biocompatible bioerosive polymer.

[31] (a) The surfactant polymer contains 0.1% (w / w) chitosan, or (b) The surfactant polymer contains 0.5-2.5% (w / w) poly(vinyl) alcohol (PVA), or (c) The surfactant polymer contains 0.5-2.5% (w / w) of polyvinylpyrrolidone (PVP), or (d) Any combination of (a) to (c), The method according to any one of embodiments 24 to 30.

[32] The method according to any one of embodiments 24 to 31, wherein the first mixture comprises 0.75% to 2% (w / w) of poly(vinyl) alcohol (PVA).

[33] Use of nanoparticles according to any one of embodiments 1 to 11 for delivery to polypeptides.

[34] Use of any one of embodiments 12 to 14 of the pharmaceutical composition for delivery to a polypeptide.

Claims

1. Nanoparticles comprising a composition containing a polypeptide, wherein the composition is encapsulated in a material containing a biocompatible, bio-erosive polymer and a surfactant polymer.

2. The nanoparticle according to claim 1, wherein the biocompatible bio-erosive polymer is polymethacrylate.

3. The nanoparticles according to claim 1, wherein the nanoparticles have a diameter of less than 300 nm.

4. The nanoparticle according to claim 1, wherein the nanoparticle contains at least 5% w / w of polypeptide.

5. The nanoparticle according to claim 1, wherein the polypeptide is IgY.

6. The nanoparticles according to claim 5, wherein IgY is obtained from highly immunized eggs.

7. The nanoparticles according to claim 1, wherein the composition is a highly immunized egg product.

8. The nanoparticles according to claim 1, wherein the composition is a defatted fraction from egg yolk containing at least 95% by weight of IgY.

9. The nanoparticles according to claim 1, wherein the composition comprises purified IgY.

10. Biocompatible, bio-erosive polymers (a) Methacrylic acid-methyl methacrylate copolymer (1:1), or (b) Methacrylic acid-methyl methacrylate copolymer (1:2), or (c) Both (a) and (b) The nanoparticles according to claim 1, comprising:

11. The nanoparticle according to claim 1, wherein the surfactant polymer is selected from chitosan, poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

12. Nanoparticles according to any one of claims 1 to 11, and Pharmacologically acceptable carriers A pharmaceutical composition containing the following:

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutically acceptable carrier is suitable for oral administration.

14. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is in a form selected from the group consisting of powder, tablets, enteric-coated tablets, capsules, enteric-coated capsules, suspensions, solutions, and oral beverages.

15. A pharmaceutical composition for delivering polypeptides, comprising nanoparticles according to any one of claims 1 to 11, A pharmaceutical composition that is formulated for oral administration.

16. (a) A step of preparing a first mixture comprising a polypeptide composition, water, and a surfactant polymer. (b) The step of ultrasonically treating the first mixture, (c) Adding a biocompatible bio-erosive polymer to ethanol and sonicating it to produce a second mixture, (d) Mixing the first mixture and the second mixture with an organic solvent to form a third mixture, (e) Homogenizing the third mixture using a probe homogenizer at a speed of approximately 5,000 to 15,000 rpm to produce a crude emulsion. (f) The crude emulsion is subjected to high-pressure homogenization using a high-pressure homogenizer at a pressure of approximately 10,000 psi to 30,000 psi to form a fourth mixture, and (g) Evaporate the ethanol and organic solvent from the fourth mixture to produce a suspension containing nanoparticles. A method for preparing nanoparticles according to claim 1, comprising:

17. The method according to claim 16, further comprising the step of centrifuging the suspension to obtain nanoparticles.

18. The method according to claim 16, wherein the surfactant polymer is selected from chitosan, poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

19. The method according to claim 16, wherein the organic solvent comprises dichloromethane.

20. The method according to claim 16, wherein the first mixture comprises about 2% to 5% (w / w) of polypeptide.

21. The method according to claim 16, wherein the polypeptide is IgY.

22. The method according to claim 16, wherein the second mixture contains a biocompatible, bioerosive polymer in an amount of 10 mg / mL to 20 mg / mL.

23. (a) The surfactant polymer contains 0.1% (w / w) chitosan, or (b) The surfactant polymer contains 0.5–2.5% (w / w) of poly(vinyl) alcohol (PVA), or (c) The surfactant polymer contains 0.5–2.5% (w / w) of polyvinylpyrrolidone (PVP), or (d) Any combination of (a) to (c), The method according to claim 16.

24. The method according to claim 16, wherein the first mixture comprises 0.75% to 2% (w / w) of poly(vinyl) alcohol (PVA).

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