Drug delivery systems and methods comprising polysialic acid and / or other polymers

Abstract

To provide particles that can access the interior of cells and procure the intracellular release of drugs.SOLUTION: There is provided a composition comprising a plurality of nanoentities comprising an inner portion surrounded with an outer shell, in which the outer shell includes a polymer and a targeting moiety and the inner portion includes at least one hydrophobic compound, the polymer being selected from the group consisting of polysialic acid and / or pegylated-polysialic acid, hyaluronic acid and / or pegylated-hyaluronic acid, polyglutamic acid and / or pegylated-polyglutamic acid, poly(aspartic acid) and / or pegylated-poly(aspartic acid), polylactic acid and / or pegylated polylactic acid, and mixtures thereof; and the hydrophobic compound being selected from the group consisting of oil, fatty acids, alkanes, cycloalkanes, bile salts, terpenoids, terpenes, fat-soluble vitamins, and surfactants.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to Spanish Patent Application No. P201731277, entitled "Sistemas de Liberacion de Farmacos de Acido Polisialico y Metodos," filed November 2, 2017. In the United States and other countries, this application is incorporated herein by reference in its entirety, where applicable.

[0002] FIELD OF THE INVENTION The present invention relates generally to particles comprising nanocapsules or nanoentities comprising polymers such as polysialic acid, which act as carriers for delivering drugs or other active substances to the interior of cells, or for other uses. [Background technology]

[0003] Targeted delivery of pharmaceuticals into the body remains a challenge, as many drugs are unable to exert their effects efficiently due to difficulty in accessing target cells.

[0004] Therefore, there is a need for improved drug delivery. Summary of the Invention

[0005] The present invention generally relates to particles, including nanocapsules or other nanoentities comprising polymers such as polysialic acid (hereinafter "PSA"), which can access the interior of cells, where they release their contents. The subject matter of the present invention, in some cases, includes one or more systems and / or articles of interrelated manufacture, alternative solutions to a particular problem, and / or multiple different uses.

[0006] The present inventors have fabricated nanoentities, such as nanocapsules, that include an inner portion surrounded by an outer shell, the outer shell comprising polysialic acid (PSA), with the PSA conjugated to a targeting moiety, specifically the cell-penetrating peptide Lyp-1 or cLyp-1. This can be seen in Example 1. The present inventors have also demonstrated that these nanocapsules can contain pharmaceuticals such as paclitaxel and docetaxel. Furthermore, the present inventors have demonstrated that the nanocapsules exhibit enhanced drug delivery into tumor tissue, resulting in greater efficacy than the pharmaceutical alone in an orthotopic lung tumor model (see Example 2). The present inventors have also demonstrated that other targeting moieties, such as CendR, can also be used (see Example 3). Example 5 describes the formulation of PSA nanocapsules conjugated with paclitaxel and other anticancer drugs. As shown in Examples 6, 7, and 13, PSA and polymers such as hyaluronic acid are conjugated to hydrophobic moieties, such as C. 16 It can be attached to an alkyl group.

[0007] The present inventors have also succeeded in producing nanocapsules conjugated with pharmaceuticals, which are monoclonal antibodies, as shown in Examples 8 to 10, where various polymers and nanocapsules, namely, PSA, PSA with tLyp-1, C 12 Alkyl-functionalized PSA, C 16 The nanocapsules were characterized, for example, in terms of their toxicity, stability, and loading capacity (see Examples 10 and 11). Furthermore, the nanocapsules were found to interact with cells and induce cellular internalization of the bound antibody, i.e., the nanocapsules were enveloped by the cell membrane and drawn into the cells, where the antibody was released (see Example 12).

[0008] Thus, in one aspect, the invention relates to a composition comprising a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell comprising a polymer and a targeting moiety, and the inner portion comprising at least one hydrophobic compound.

[0009] In another aspect, the invention relates to a composition comprising a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell comprising a polymer, and the inner portion comprising at least one hydrophobic compound, with the proviso that at least about 90% of the polymer is not hyaluronic acid.

[0010] In another aspect, the invention relates to a composition comprising a plurality of nano-entities for use as a medicament.

[0011] In one aspect, the present invention generally relates to compositions. In one set of embodiments, the compositions include a plurality of nano-entities, e.g., nanocapsules, comprising an inner portion (or core) surrounded by an outer shell. In some cases, the outer shell comprises a polymer, such as PSA. The inner portion comprises at least one hydrophobic compound.

[0012] In some embodiments, the shell comprises a targeting moiety, i.e., a molecule that enables targeting or selective targeting of the nanostructure. In certain embodiments, the shell comprises a cell-penetrating and / or tumor / tissue-penetrating peptide. In some cases, the targeting moiety and / or cell-penetrating peptide and / or tumor / tissue-penetrating peptide are chemically bound to PSA.

[0013] In another set of embodiments, the composition comprises a plurality of nanocapsules comprising an interior portion surrounded by an outer shell. In some embodiments, the outer shell comprises PSA and a targeting moiety chemically linked to the PSA. In some cases, the targeting moiety has the sequence Z 1 X 1 X 2 Z 2 and Z 1 is R or K, and Z 2 is R or K, and X 1 and X 2are each an amino acid residue. In some cases, the peptide includes the sequence RGD or the sequence NGR. For example, the peptide includes the sequence J 1 R.G.D., J. 1 RGDJ 2 , R.G.D.J. 2 , J 1 N.G.R., J. 1 NGRJ 2 , NGRJ 2 (These abbreviations, K, R, N, G, D, etc., are standard single-letter codes for amino acid residues used by those skilled in the art; see below for details.) In some cases, the targeting moiety is Z 1 X 1 X 2 Z 2 Both the RGD sequence and the iRGD sequence (e.g., iRGD peptide) or Z 1 X 1 X 2 Z 2 The present invention also includes peptides having both the iNGR sequence and the NGR sequence (e.g., iNGR).

[0014] In another set of embodiments of another aspect, the composition comprises a plurality of nanoentities comprising an inner portion surrounded by an outer shell, the outer shell comprising a polymer such as PSA, and at least some of the nanoentities further comprising a monoclonal antibody contained within the inner portion.

[0015] In another embodiment, the composition comprises an inner portion surrounded by an outer shell, the outer shell comprising PSA and a targeting moiety chemically linked to the PSA, the targeting moiety comprising the sequence Z 1 X 1 X 2 Z 2 and / or a peptide having the sequence RGD and / or the sequence NGR, wherein Z1 is R or K, and Z 2 is R or K, and X 1 and X 2 is an amino acid residue.

[0016] In another set of embodiments of another aspect, the composition comprises an entity having a maximum average diameter of less than about 1 micrometer. The entity has a surface that, in some embodiments, comprises a polymer, such as PSA, and a targeting moiety. In some cases, the entity is not a liposome (see below for a discussion of liposomes).

[0017] Another set of embodiments generally relates to compositions comprising a plurality of nano-entities, e.g., nanocapsules, comprising an interior portion surrounded by an outer shell. The outer shell comprises a polymer, such as PSA, optionally bound to a hydrophobic moiety, e.g., by covalent bonding, electrostatic bonding, etc. The interior portion, in certain cases, comprises at least one hydrophobic compound. In some embodiments, the outer shell comprises a polymer, such as PSA, a targeting moiety, and a hydrophobic moiety. In some cases, at least a portion of the PSA is bound to the targeting moiety and / or the hydrophobic moiety. In some embodiments, the hydrophobic moiety is a C2-C 24 or C 12 and the like alkyl groups.

[0018] In another embodiment, the composition comprises a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell comprising PSA and a targeting moiety comprising a cell-penetrating peptide chemically bound to the PSA.

[0019] In another set of embodiments of another aspect, the composition includes a plurality of nano-entities, e.g., nanocapsules, comprising an interior portion surrounded by an outer shell. In some cases, the outer shell consists essentially of a polymer, such as PSA. In certain cases, the interior portion comprises at least one hydrophobic compound.

[0020] In one aspect, the composition comprises a plurality of nanoentities comprising an interior portion surrounded by an outer shell, the outer shell comprising hyaluronic acid, and at least some of the nanoentities further comprising a monoclonal antibody.

[0021] In another embodiment, the composition comprises an inner portion surrounded by an outer shell, the outer shell comprising a plurality of nano-entities comprising PGA and / or PASP and a targeting moiety.

[0022] In yet another embodiment, the composition comprises an inner portion surrounded by an outer shell, the outer shell comprising PGA and / or PASP and a targeting moiety, the targeting moiety comprising the sequence Z 1 X 1 X 2 Z 2 and / or a peptide having the sequence RGD and / or the sequence NGR, Z 1 is R or K, and Z 2 is R or K, and X 1 and X 2 is an amino acid residue.

[0023] In yet another embodiment, the composition comprises a plurality of nanoentities comprising an inner portion surrounded by an outer shell, the outer shell comprising PGA and / or PASP, and at least some of the nanoentities further comprising a monoclonal antibody contained within the inner portion.

[0024] In one aspect, the composition comprises a plurality of nano-entities comprising an interior portion surrounded by an outer shell, the outer shell comprising hyaluronic acid associated with a hydrophobic moiety.

[0025] In another aspect, the composition comprises a plurality of nanoentities comprising an interior portion surrounded by an outer shell, the outer shell comprising a polymer selected from the group consisting of a polyacid, a polyester, a polyamide, or a mixture thereof, and at least some of the nanoentities further comprising a monoclonal antibody.

[0026] In yet another embodiment, the composition comprises a plurality of nanoentities comprising an inner portion surrounded by an outer shell, the outer shell comprising hyaluronic acid bound to a hydrophobic moiety, and at least some of the nanoentities further comprising a small molecule have a molecular weight of less than 1000 Da.

[0027] In another set of embodiments, the composition is a pharmaceutical composition.

[0028] Further embodiments of the present invention generally relate to the use of any of the compositions described above or any of the compositions described herein as a drug.Furthermore, some embodiments of the present invention generally relate to the method of administering any of the compositions described above or any of the compositions described herein to an organism, such as a human.In some cases, the organism is a subject with cancer or other diseases.For example, any of the compositions described above (or any of the compositions described herein) can further comprise a suitable therapeutic agent, such as an anti-cancer agent or antibody.

[0029] Another aspect of the invention generally relates to a method. In some embodiments, the method comprises reacting a carboxylate moiety on PSA with an aminoalkyl(C1-C4)maleimide and / or aminoalkyl(C1-C4)methacrylamide, and reacting the resulting aminoalkyl(C1-C4)maleimide and / or aminoalkyl(C1-C4)methacrylamide with a thiol group (e.g., derived from a cysteine ​​group) on the targeting moiety to obtain a PSA-aminoalkyl(C1-C4)succinimide-peptide and / or PSA-aminoalkyl(C1-C4)amide-isopropyl-peptide composition. In some embodiments, the method comprises reacting a carboxylate moiety on PSA with an activating agent, such as N-hydroxysuccinimide, triazine, or carbodiimide, and reacting the formed intermediate with an amino group (e.g., derived from a lysine or arginine group) on the targeting moiety to obtain a PSA-amide-peptide composition.

[0030] Disclosed herein are several methods of administering compounds to a subject for the prevention or treatment of a particular condition. In each such aspect of the invention, it should be understood that the invention specifically includes the compound for use in the treatment or prevention of that particular condition, and also the use of the compound in the manufacture of a medicament for the treatment or prevention of that particular condition.

[0031] In another aspect, the invention encompasses methods of making one or more of the embodiments described herein, e.g., nanocapsules. In yet another aspect, the invention encompasses methods of using one or more of the embodiments described herein, e.g., nanocapsules.

[0032] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings.

[0033] Non-limiting embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, which are schematic and not intended to be to scale. In the drawings, identical or nearly identical components shown will typically be represented by a single numeral. For purposes of clarity, not every component will be shown in every drawing, nor will every component of each embodiment of the present invention shown, unless illustration is necessary for those skilled in the art to understand the invention. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 shows the coupling reaction of sialic acid with a peptide that acts as a targeting moiety. [Figure 2] 2A-2B show data demonstrating delivery of nanocapsules to mice according to certain embodiments of the present invention. [Figure 3] FIG. 1 shows a comparison of the delivery of certain nanocapsules described herein with Abraxane® (nab-paclitaxel). [Figure 4] FIG. 10 shows the weight change in mice treated with specific nanocapsules according to yet another embodiment of the present invention. [Figure 5] 5A-5B show the in vivo effects of certain nanocapsules according to another embodiment of the present invention. [Figure 6] FIG. 1 shows a method for making a modified PSA according to another embodiment of the present invention. [Figure 7]FIG. 1 shows the cytotoxicity of various polymer nanocapsules according to another embodiment of the present invention. [Figure 8] 8A-8D show the effect of polymer nanocapsule delivery to cells, according to one embodiment of the present invention. [Figure 9] 9A-9B show the stability of various mAb-loaded polymer nanocapsules as measured by DLS in another embodiment of the present invention. [Figure 10] 10A-10C show the stability of various mAb-loaded polymer nanocapsules as measured by NTA in yet another embodiment of the present invention. [Figure 11] FIG. 10 shows positive cells incubated with various polymer nanocapsules according to yet another embodiment of the present invention. [Figure 12] 12A-12B show cells loaded with nanocapsules according to yet another embodiment of the present invention. [Figure 13] 13A-13C show 1H-NMR spectra of PSA, tLyp1, and the conjugate PSA-tLyp1 of certain embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0035] (Detailed Description of the Invention) The present invention generally relates to particles, including nanocapsules or other nanoentities comprising polymers such as polysialic acid (PSA). The particles can access the interior of cells and / or cause intracellular release of attached drugs. In one aspect, the present invention relates to nanocapsules or other entities having an exterior or surface comprising a polymer such as PSA. In some cases, a targeting moiety, such as Lyp-1 or tLyp-1 peptide, is attached to the polymer using, for example, an aminoalkyl(C1-C4) succinimide or other linker. These are generated, for example, by reacting a carboxylate moiety on the polymer with an aminoalkyl(C1-C4) maleimide or aminoalkyl(C1-C4) methacrylamide, followed by reaction of the resulting aminoalkyl(C1-C4) maleimide or aminoalkyl(C1-C4) methacyrlamide with a cysteine ​​or other sulfur group. For example, targeting moieties are attached to the polymer by reacting a carboxylate moiety on the polymer with N-hydroxysuccinimide or a carbodiimide and reacting the resulting intermediate with a lysine or arginine group on the targeting peptide to yield a polymer-amide-peptide. Other aspects of the invention relate generally to methods of making or using such compositions, kits containing such compositions, and the like.

[0036] Uses of Entities In one aspect, the present invention generally relates to particles or other entities comprising polymers such as PSA. Such particles or entities are used, for example, in drug delivery applications. For example, such particles are delivered into a subject to reach a tumor afflicting the subject. Delivery of the particles into tumor cells is facilitated by a targeting moiety, e.g., a cell- or tissue-penetrating peptide such as Lyp-1 or tLyp-1, or other peptides discussed herein (e.g., CendR peptides). Other peptide, antibody (e.g., full-length antibody, nanobody, single-chain variable fragment, etc.), or aptamer targeting moieties are also used in certain embodiments, e.g., as discussed herein. After delivery, the particles can access target cells, e.g., tumor cells, and release a drug (e.g., a therapeutic agent or anticancer drug) contained therein. To date, particles or other entities comprising modified PSA with a targeting moiety have not been used for selective and intracellular drug release.

[0037] In some cases, the entity is present in a pharmaceutically acceptable carrier, as discussed herein, e.g., the entity is suspended in a liquid or gel, e.g., for administration to a subject. The entity can be substantially solid or define an interior space, e.g., as in a capsule. The entity is also, in some embodiments, a micelle or liposome, although in certain cases, the entity is not a liposome, as discussed herein.

[0038] Entities - Nanoentities "Entity" includes, for example, capsules, particles, and micelles. In some cases, the entity is a nanoentity. As used herein, a "nanoentity" is typically an entity having an average diameter of less than 1,000 nm, e.g., less than 750 nm, less than 500 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, or less than 100 nm. In some cases, the entity has an average diameter of at least 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, or 1,000 nm. Combinations of any of the above diameters are also possible, for example, entities having average diameters ranging from 100 nm to 300 nm, 1,000 nm to 1 nm, 1,000 nm to 10 nm, 750 nm to 1 nm, 500 nm to 10 nm, 300 nm to 10 nm, 250 nm to 10 nm, 200 nm to 10 nm, 150 nm to 10 nm, 100 nm to 10 nm, etc. In some embodiments, more than one entity is present, and in such cases, the average (arithmetic mean) diameter of the multiple entities has the dimensions described herein. In some cases, entities have a range of diameters. Such entities are determined by various methods, such as dynamic or laser light scattering. Non-limiting examples of nanoentities include nanoparticles, nanocapsules, micelles, or other entities, such as those described herein. Such nanoentities, in some cases, have the dimensions described in this paragraph.

[0039] In some cases, the entity includes an interior portion surrounded by an outer shell, e.g., an outer shell exposed to the environment surrounding the entity. The interior portion is symmetrically or asymmetrically located within the entity. The interior portion includes, for example, a liquid (e.g., non-aqueous or aqueous), a solid, and / or a combination thereof. In some embodiments, the interior portion includes one or more pharmaceutical agents or drugs, e.g., any of those described herein. For example, the interior portion includes a monoclonal antibody or a small molecule, e.g., docetaxel. In some cases, the interior portion (including the portion it contains) is protected from exposure to the external environment, e.g., by the outer shell.

[0040] Entities - Capsules / Nanocapsules, Particles / Nanoparticles In some cases, the entity is a capsule (e.g., a nanocapsule). A capsule may be substantially solid or have a comb-like or gel-like shell. Additionally, in some cases, the entity is a particle, such as a nanoparticle. A particle is solid and has a defined shape. In some cases, a particle is an entity having an interior portion surrounded by an outer shell, e.g., a particle is a capsule. Nanocapsules are in the nanometer range in size. When a nanoparticle is roughly spherical, it may be referred to as a nanosphere. Nanocapsules are substantially uniform but have other surface features, such as targeting moieties, penetration enhancers, antibodies, etc., including those described herein.

[0041] In some cases, a particle is an entity having an inner portion surrounded by an outer shell, for example, the particle is a capsule or nanocapsule. In some cases, a nanocapsule has a size in the nanometer range, including an inner core and an outer shell having a composition distinguishable from the inner core. The inner core can be, for example, a liquid or solid material. In many cases, but not always, the inner core is oil. The outer shell is formed from a continuous material and is usually not covalently bonded to the inner core. In some cases, the outer shell has an average thickness of at least 1 nm, at least 2 nm, at least 3 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, or at least 200 nm.

[0042] In some cases, the nano-entity comprises only one outer shell.

[0043] Entities - Micelles In some cases, the entity is a micelle. Micelles are typically formed from multiple surfactant or amphiphilic molecules that define an interior and an exterior surface. For example, the surfactant molecules are arranged to have a relatively hydrophilic exterior and a relatively hydrophobic interior, formed, for example, from a monolayer of surfactant or amphiphilic molecules. In some cases, micelles are in the nanometer range in size. In some embodiments, micelles are formed by amphiphilic molecules at a concentration above the critical micelle concentration (CMC) when dispersed in an external phase. When the external liquid phase is aqueous, the hydrophilic portions of the amphiphilic molecules face toward the external phase. Depending on the concentration of the amphiphilic molecules, micelles may self-organize to form larger structures called clusters of micelles. Micelles are formed, for example, from surfactant molecules with their hydrophilic portions on the surface and their hydrophobic portions facing inward (or vice versa in some cases).

[0044] Entities - Liposomes Liposomes can have similar structures but are typically formed from a bilayer of surfactant or amphiphilic molecules (e.g., a lipid bilayer) that defines an interior, middle, and outer shell, e.g., the interior is relatively hydrophilic, the middle (e.g., the outer shell of the liposome, formed by the surfactant or amphiphilic molecule bilayer) is relatively hydrophobic, and the exterior of the liposome is an aqueous or hydrophilic environment.

[0045] As used herein, the property of being "hydrophilic" is understood to be the essential property of a molecule or functional group to enter or remain in the aqueous phase. Accordingly, the property of being "hydrophobic" is understood to be the essential property of a molecule or functional group to exhibit an outward behavior toward water, i.e., to not enter water or to exhibit a tendency to leave the aqueous phase. For further details, see Rompp Lexikon Lacke und Druckfarben, Georg Thieme Verlag, Stuttgart, NY, 1998, "Hydrophilicity", "Hydrophobicity", pages 294 and 295. In some cases, a hydrophilic (or water-soluble) entity is one whose log P is less than 1.5, and a hydrophobic (or liposoluble) entity is one whose log P is greater than 1.5, where log P is the octanol-water partition coefficient of the entity.

[0046] When the inner portion is present within an entity, the inner portion contains a liquid, and in some cases, the liquid is aqueous or non-aqueous. In some cases, the liquid includes a saline or salt-water solution. The liquid may optionally contain a drug or other pharmaceutical agent, for example, for delivery to a subject. Non-limiting examples of drugs or other pharmaceutical agents are discussed herein. For example, the inner portion includes a monoclonal antibody or a small molecule, such as docetaxel.

[0047] In some embodiments, the nanoentity comprises an outer shell consisting essentially of a single layer of material comprising a polymer, such as PSA. In other embodiments, the nanoentity comprises a single shell comprising a polymer, such as PSA. In other embodiments, the outer shell comprises multiple layers, one of which comprises a polymer, such as PSA. In further embodiments, the layer comprising the polymer is the outermost layer.

[0048] In some embodiments, the interior portion of a nano-entity, e.g., a nanocapsule, nanoparticle, micelle, or liposome, comprises a solid, a semi-solid (e.g., a gel), a liquid, a gas, or a combination thereof. The interior portion is aqueous or non-aqueous, or comprises both aqueous and non-aqueous portions. In some embodiments, the interior portion comprises one or more pharmaceutical agents, drugs, etc.

[0049] In other embodiments, the inner portion comprises a non-aqueous portion. In further embodiments, the non-aqueous portion is a non-aqueous liquid. In further embodiments, the non-aqueous liquid comprises a hydrophobic compound, for example, an oil. In further embodiments, the non-aqueous liquid comprises an oil and a surfactant. In further embodiments, the inner portion comprises a fatty acid. In further embodiments, the inner portion comprises a monoglyceride. In further embodiments, the inner portion comprises a diglyceride. In further embodiments, the inner portion comprises a triglyceride. In further embodiments, the inner portion comprises a medium chain triglyceride. In further embodiments, the inner portion comprises a long chain triglyceride.

[0050] Hydrophobic compounds When the interior portion of the entity (e.g., a capsule, particle, micelle, or other nano-entity, such as those discussed herein) is non-aqueous, the non-aqueous liquid forming the interior portion comprises one or more hydrophobic compounds selected from, for example, oils, fatty acids, alkanes, cycloalkanes, bile salts, bile salt derivatives, terpenoids, terpenes, terpene-derived moieties, and fat-soluble vitamins, and / or at least one surfactant. These oils can be selected from natural, semi-synthetic, and synthetic oils used in pharmaceuticals, such as oils derived from plant or animal sources, hydrocarbon oils, or silicone oils. Oils suitable for practicing certain embodiments of the present invention include, but are not limited to, mineral oil, squalene oil, flavor oils, silicone oils, essential oils, water-insoluble vitamins, isopropyl stearate, butyl stearate, octyl palmitate, cetyl palmitate, tridecyl behenate, diisopropyl adipate, dioctyl sebacate, menthyl anthranilate, cetyl octanoate, octyl salicylate, isopropyl myristate, neopentyl glycol ketol dicaprate, decyl oleate, and C lactate. 12 ~C 15Alkyl, cetyl lactate, lauryl lactate, isostearyl neopentanoate, myristyl lactate, isocetyl stearoyl stearate, octyldodecyl stearoyl stearate, hydrocarbon oil, isoparaffin, liquid paraffin, isododecane, petroleum jelly, argan oil, rapeseed oil, chili oil, coconut oil, corn oil, cottonseed oil, linseed oil, grape seed oil, mustard oil, olive oil, palm oil, fractionated palm oil, peanut oil, castor oil, pine nut oil, mustard oil, pumpkin seed oil, rice bran oil, safflower oil, tea tree oil, Truffle oil, vegetable oil, apricot kernel oil, jojoba oil, macadamia nut oil, wheat germ oil, almond oil, soybean oil, sesame seed oil, hazel oil, sunflower oil, hemp seed oil, rosewood oil, kukui nut oil, avocado oil, walnut oil, fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, sage leaf oil, eucalyptus leaf oil, lemon leaf oil, melaleuca leaf oil, oregano oil, patchouli leaf oil, peppermint leaf oil, pine leaf oil, Rosemary leaf oil, spearmint oil, tea tree leaf oil, thyme oil, flower essential oils, chamomile oil, clary sage oil, clove oil, geranium flower essential oil, hyssop flower essential oil, jasmine oil, lavender oil, mauka flower essential oil, marjoram flower essential oil, orange flower essential oil, rose flower essential oil, ylang-ylang flower essential oil, bark oil, cassia bark oil, cinnamon bark oil, sassafras bark oil, tung oil, camphor wood oil, cedarwood oil, rosewood oil, sandalwood oil, ginger oil, tall oil, castor oil, myrrh oil, fruit peel oil, bergamot peel oil, grapefruit peel oil, lemon Peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, oleic acid, linoleic acid, oleyl alcohol, isostearyl alcohol, ethyl oleate, medium chain triglycerides such as mixtures of decanoyl and octanoyl glycerides (Miglyol® 810N, Miglyol® 812N, Kollisolv® MCT, Captex® 300, Captex® 355, Labrafac® Lipophile WL1349), Labrafil® M 2125 CS (linoleoyl macrogol-6 glycerides), Labrafil® M 2130CS (lauroyl macrogol-6 glycerides), Labrafil® M 1944 CS (oleoyl polyoxyl-6 glycerides), Labrafac® PG (propylene glycol dicaprylocaprate), Rylo® (mixture of fatty acids), Peceol® (glycerol monooleate) and Maisine® (glycerol monolinoleate), as well as their synthetic or semi-synthetic derivatives, and combinations thereof.

[0051] In some cases, the oil is one or more of peanut oil, cottonseed oil, olive oil, castor oil, soybean oil, safflower oil, sesame oil, corn oil, palm oil, alpha-tocopherol (vitamin E), isopropyl myristate, squalene, Miglyol®, Labrafil®, Labrafac®, Peceol®, Captex®, Kollisolv® MCT, and Maisine®, or mixtures thereof. Other suitable oils include those from the terpene family formed by isoprene units (2-methylbuta-1,3-diene) and subdivided according to their carbon atoms: hemiterpenes (C5), monoterpenes (C6), hemiterpenes (C7), hemiterpenes (C8), hemiterpenes (C9), hemiterpenes (C10), hemiterpenes (C11), hemiterpenes (C12), hemiterpenes (C13), hemiterpenes (C14), hemiterpenes (C15), hemiterpenes (C16), hemiterpenes (C17), hemiterpenes (C18), hemiterpenes (C19 ... 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), sesterterpenes (C 25 ), triterpenes (C 30 ), tetraterpenes (C 40 Examples of suitable oils include oils derived from cereals such as cereals like cereals, dairy products, and dairy products (e.g., cereals containing gluten, dairy products, and dairy products), vitamin A, squalene, and the like. In some embodiments, the non-aqueous liquid forming the inner portion may contain water-insoluble stabilizers, preservatives, surfactants, organic solvents, and mixtures thereof to maximize the stability of the formulation. Combinations of one or more of these and / or other oils are also possible in various embodiments.

[0052] When the interior portion of an entity (e.g., a capsule, particle, micelle, or other nano-entity, such as those discussed herein) is aqueous, the aqueous liquid forming the interior portion can, in certain embodiments, consist of water containing at least one salt.

[0053] Additionally, in some embodiments, the aqueous liquid forming the inner portion may contain one or more water-soluble stabilizers, preservatives, surfactants, glycols, polyols, sugars, thickeners, gelling agents, and combinations thereof, and / or other suitable excipients used, for example, to improve the stability of the formulation, to adjust the viscosity of the final composition, to control the release rate from the internal aqueous phase, etc.

[0054] polymer In one set of embodiments, an entity (e.g., a capsule, particle, micelle, or other nano-entity, such as those discussed herein) comprises a polymer, such as a PSA. The polymer is distributed evenly throughout the entity or concentrated in specific regions of the entity, such as the outer shell of a capsule or other outer surface of the entity. In some cases, at least 50% by weight of a portion of the entity, such as the shell, comprises the polymer, and in certain cases, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% by weight of the portion of the entity comprises the polymer. In some cases, a portion of the entity may consist essentially of the polymer.

[0055] Certain embodiments of the present invention utilize various polymers. For example, the polymer, in one set of embodiments, is a polyacid, poly(amino acid), or polyester. Non-limiting examples of these polymers include PSA, hyaluronic acid (HA), polyglutamic acid (PGA), PEGylated polyglutamic acid (PGA-PEG), poly(aspartic acid) (PASP), PEGylated polyaspartic acid (PASP-PEG), polylactic acid, PEGylated polylactic acid (PLA-PEG), PEGylated poly(lactic-co-glycolic acid) (PLGA-PEG), polyasparagine, PEGylated polyasparagine, alginic acid, PEGylated alginic acid, polymalic acid, PEGylated polymalic acid, and the like. Certain embodiments utilize combinations of these and / or other polymers. For example, such polymers are used to form nanoentities, e.g., containing monoclonal antibodies or small molecules contained within the interior portion of the nanoentity, or for other applications, such as those described herein.

[0056] Polymer - Polysialic Acid, PSA In one set of embodiments, the polymer comprises PSA. PSA is generally composed of multiple sialic acid units, often linked together via 2-->8 and / or 2-->9 linkages to form the polymer, although other linkage arrangements are possible. Typically, there are at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500 sialic acid units linked together to form PSA. In some cases, the PSA has 1000 or fewer, 500 or fewer, 200 or fewer, 100 or fewer, 50 or fewer, 30 or fewer, or 10 or fewer sialic acid units linked together to form PSA. Any combination of these is also possible; for example, PSA has 2 to 100 sialic acid units linked together. It should be noted that the sialic acid units do not have to be identical and can be independently the same or different even within the same PSA molecule. It should also be noted that PSA does not necessarily have to be a linear (straight-chain) chain; various branching arrangements are also possible. For example, a sialic acid unit may be linked to three or more different sialic acid units, thereby creating branch points within the PSA molecule.

[0057] By way of non-limiting example, PSA can have a variety of molecular weights, such as 4 kDa, 30 kDa, 95 kDa, etc. In some cases, PSA contains more than 300 sialic acid units. By way of further non-limiting example, PSA can have a molecular weight of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. In some cases, the PSA has a molecular weight of 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 10 kDa or less, 5 kDa or less, 3 kDa or less, or 1 kDa or less, or any combination thereof, such as a PSA having a molecular weight of about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, or about 10 kDa to about 50 kDa (unless otherwise specified, molecular weights described herein are number average molecular weights).

[0058] It should also be noted that the polysialic acids need not necessarily be identical. For example, in some embodiments, PSA has a varying number of sialic acid units and / or different sialic acid units are present in the various PSA molecules present. In some cases, one or several types of PSA molecules may be present, e.g., one or more forms comprise at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more of the PSA molecules present, i.e., on a molar basis.

[0059] Non-limiting examples of sialic acid units present in PSA include, but are not limited to, N-acetylneuraminic acid (Neu), 2-keto-3-deoxynonic acid (Kdn), lactamic acid, N-sialic acid, and / or O-sialic acid. Other examples include N-glycolylneuraminic acid (Neu5Gc), 9-O-acetyl-8-O-methyl-N-acetylneuraminic acid (Neu5,9Ac28Me), and 7,8,9-tri-O-acetyl-N-glycolylneuraminic acid (Neu5Gc7,8,9Ac3). "Sia" generally represents an unspecified sialic acid unit. In some embodiments, the sialic acid unit comprises any derivative of neuraminic acid (a nine-carbon sugar), including the 43 derivatives commonly found in nature. Such amino acids include, but are not limited to, Neu; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu5,7,8,9Ac4; Neu5Ac9Lt; Neu4,5Ac29Lt; Neu5Ac8Me; Neu5,9Ac28Me; Neu5Ac8S; Neu5Ac9P; Neu2en5Ac; Neu2en5,9Ac2; Neu2en5Ac9Lt; Neu2,7an5Ac; Neu5Gc; Neu4A and Knd9Ac. In one set of embodiments, the sialic acid units (prior to polymerization to form PSA) can each independently have the following structure: [ka] R 1is H; an alpha bond to Gal(3 / 4 / 6), GalNAc(6) (N-acetylgalactosamine), GlcNAc(4 / 6), Sia(8 / 9), or 5-O-Neu5Gc; an oxygen attached to C-7 in a 2,7-anhydro molecule; or an anomeric hydroxyl (double bond to C-3) that is eliminated with Neu2en5Ac. 2 is H; an alpha bond to Gal(3 / 4 / 6), GalNAc(6), GlcNAc(4 / 6), Sia(8 / 9), or 5-O-Neu5Gc; an oxygen bonded to C-7 in a 2,7-anhydro molecule; or an anomeric hydroxyl (double bond to C-3) that is eliminated with Neu2en5Ac. 4 is H; -acetyl; anhydro at C-8; Fuc (fucose); or Gal (galactose). 5 is amino; N-acetyl; N-glycolyl; hydroxyl; N-acetimidoyl; N-glycolyl-O-acetyl; N-glycolyl-O-methyl; or N-glycolyl-O-2-Neu5Gc. 7 is H; -acetyl; anhydro at C-2; or is substituted by amino and N-acetyl in Leg (legionaminic acid). 8 is H; -acetyl; anhydro at C-4; -methyl; -sulfate; Sia (sialic acid); or Glc (glucose). 9 is H; -acetyl; -lactyl; -phosphate; -sulfate; Sia; or OH substituted by H in Leg. In some cases, PSA is colominic acid (when only 2-->8 bonds are present).

[0060] As used herein, sialic acid includes water-soluble salts and water-soluble derivatives of sialic acid.For example, sialic acid salts are sodium salts, potassium salts, magnesium salts, calcium salts, or zinc salts.In one embodiment, at least a portion of sialic acid exists as sodium salts.A combination of multiple types of sialic acid can be used, for example, as a subunit of PSA and / or as PSA of different molecules.

[0061] In one set of embodiments, at least a portion of the sialic acids in the PSA are modified (although it should be understood that in other embodiments, the PSA is not necessarily modified). For example, in some cases, one or more sialic acid units are modified by conjugation with, for example, polyethylene glycol, an alkyl, or other hydrophobic moiety. Hydrophobic moieties include hydrophobic molecules or portions thereof, such as alkyl groups, such as those discussed herein.

[0062] In some embodiments, the nanoentity does not comprise polyarginine or protamine.

[0063] However, it should be understood that other polymers may be used in addition to and / or in place of, for example, a PSA.

[0064] Polymer - Hyaluronic Acid, HA In one set of embodiments, the polymer comprises hyaluronic acid, a linear polymer containing repeating disaccharide structures formed by alternating additions of D-glucuronic acid and DN-acetylglucosamine linked by alternating beta-1,4 and beta-1,3 glycosidic linkages, as shown in the formula below: [ka] where the integer n represents the degree of polymerization, i.e., the number of disaccharide units in the hyaluronic acid chain. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Any combination of these is also possible, e.g., n is between 2 and 100. It should be noted that the hyaluronic acid units need not be identical and can be independently the same or different even within the same hyaluronic acid chain. It should also be noted that hyaluronic acid does not necessarily have to be a linear (straight-chain) chain; various branched configurations are possible.

[0065] Therefore, hyaluronic acid of a wide range of molecular weights can be used.High molecular weight hyaluronic acid is commercially available, while low molecular weight hyaluronic acid can be obtained by, for example, using hyaluronidase enzyme to fragment high molecular weight hyaluronic acid.By way of non-limiting example, hyaluronic acid has a variety of molecular weights, such as 4 kDa, 30 kDa, 95 kDa, etc.For example, hyaluronic acid has a molecular weight of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. In some cases, the hyaluronic acid has a molecular weight of 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 10 kDa or less, 5 kDa or less, 3 kDa or less, or 1 kDa or less, or any combination thereof is also possible, for example, the hyaluronic acid has a molecular weight of about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, or about 10 kDa to about 50 kDa.

[0066] Hyaluronic acid as used herein also includes its conjugate base (hyaluronate). This conjugate base can be an alkali salt of hyaluronic acid, including inorganic salts such as sodium salt, potassium salt, calcium salt, ammonium salt, magnesium salt, aluminum salt, and lithium salt, and organic salts such as basic amino acid salts at neutral pH. In some cases, the salt is pharmaceutically acceptable. In one embodiment, the alkali salt is the sodium salt of hyaluronic acid. Also, a combination of multiple types of hyaluronic acid can be used, for example, as a subunit of a hyaluronic acid chain and / or as different molecular hyaluronic acids.

[0067] Therefore, hyaluronic acid does not necessarily have to be the same.For example, in some embodiments, hyaluronic acid has different numbers of hyaluronic acid units (such as those described above) and / or different hyaluronic acid units exist in the different hyaluronic acid chains that exist.In some cases, one or more types of hyaluronic acid molecules exist, for example, one or more forms comprise the hyaluronic acid molecules that exist, i.e., on a molar basis, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.

[0068] In some embodiments, at least a portion of the hyaluronic acid units are modified (although it should be understood that in other embodiments, the hyaluronic acid is not necessarily modified). For example, in some cases, one or more hyaluronic acid units are modified by conjugation with, for example, polyethylene glycol, alkyl, or other hydrophobic moieties. Hydrophobic moieties include hydrophobic molecules or portions thereof, such as alkyl groups, such as those discussed herein.

[0069] Polymer - Polyglutamic Acid, PGA In another set of embodiments, the polymer comprises polyglutamic acid (PGA), a hydrophilic, biodegradable polymer composed of negatively charged glutamic acid units, which may be represented by the following formula: [ka] In the formula, the integer n represents the degree of polymerization, i.e., the number of glutamic acid units. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Any combination of these is also possible, e.g., n is 2-100. It should be noted that the hyaluronic acid glutamic acid units need not be identical and can independently be the same or different even within the same polyglutamic acid. Examples of such glutamic acid units include those discussed below. It should also be noted that the glutamic acid units do not necessarily need to be in a linear (straight-chain) chain; various branched arrangements are possible.

[0070] Thus, polyglutamic acid having a wide range of molecular weights can be used. By way of non-limiting example, polyglutamic acid can have a variety of molecular weights, such as 4 kDa, 30 kDa, 95 kDa, etc. For example, polyglutamic acid can have a molecular weight of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. In some cases, the hyaluronic acid has a molecular weight of 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 10 kDa or less, 5 kDa or less, 3 kDa or less, or 1 kDa or less, including any combination thereof, such as polyglutamic acid having a molecular weight of about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, or about 10 kDa to about 50 kDa.

[0071] As used herein, polyglutamic acid (or PGA) includes, but is not limited to, its conjugate base (glutamate) and / or water-soluble salts of PGA, such as ammonium salts, and metal salts of PGA, such as lithium, sodium, potassium, magnesium, and the like. In one embodiment, PGA includes, for example, poly-D-glutamic acid, poly-L-glutamic acid, L-glutamic acid, poly-D acid, poly-glutamic acid, poly-D-glutamic acid, poly-glutamic acid and poly-alpha-L-glutamic acid, poly-alpha-D acid, L-glutamic acid, poly-gamma-D-glutamic acid, poly-gamma-L-glutamic acid and poly-gamma-D, L-glutamic acid, and mixtures thereof. In another embodiment, PGA is present as poly-L-glutamic acid. In some cases, PGA is present as the sodium salt of poly-L-glutamic acid. In another embodiment, PGA is present as poly-alpha-glutamic acid. In yet another embodiment, the PGA is present as the sodium salt of poly-α-glutamic acid. As noted above, combinations of multiple types of polyglutamic acid may be used, for example, as subunits of the polyglutamic acid chain and / or as polyglutamic acid of different molecules.

[0072] Thus, the polyglutamates need not necessarily be identical. For example, in some embodiments, the polyglutamates vary in the number of glutamic acid units (such as those described above) and / or different polyglutamates are present in the different polyglutamic acid chains present. In some cases, one or more types of polyglutamates molecules are present, e.g., one or more forms comprise at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more of the polyglutamates present, i.e., on a molar basis.

[0073] In some embodiments, at least a portion of the polyglutamic acid units are modified (although it should be understood that in other embodiments, the polyglutamic acid is not necessarily modified). For example, in some cases, one or more polyglutamic acid units are modified, such as by attachment to polyethylene glycol, alkyl, or other hydrophobic moieties. Hydrophobic moieties include hydrophobic molecules or portions thereof, such as alkyl groups, such as those discussed herein.

[0074] Polymer - Poly(ethylene glycol), PEG In one set of embodiments, the polymer comprises poly(ethylene glycol) (PEG). In some cases, PEG is conjugated with PGA, for example, to form polyglutamic acid-polyethylene glycolic acid copolymer (PGA-PEG). However, in other cases, PEG is present, i.e., not conjugated to PGA.

[0075] Polyethylene glycol (PEG), in its most common form, has the formula: H-(O-CH2-CH2) n -OH where n is an integer representing the degree of PEG polymerization. To form the conjugate PGA-PEG, one or both of the two terminal hydroxyl groups are modified. The modified PEG is, for example, as follows: X 1 -(O-CH2-CH2) n -X 2 In the formula, X 1is a hydrogen or hydroxyl protecting group that blocks an OH radical group for subsequent reaction. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Combinations of any of these are also possible, e.g., n is 2-100.

[0076] Protecting groups for hydroxyl radicals are well known in the art, and representative (oxygen-containing) protecting groups include, for example, silyl ethers, such as trimethylsilyl ether, triethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, triisopropylsilyl ether, diethylisopropylsilyl ether, triethyldimethylsilyl ether, triphenylsilyl ether, di-tert-butylmethylsilyl ether, and the like; alkyl ethers, such as methyl ether, tert-butyl ether, benzyl ether, p-methoxybenzyl ether of 3,4-dimethoxybenzyl ether, triethyl ether, alkyl ethers, such as methyl ether, tert-butyl ether, benzyl ether, 3,4-dimethoxybenzyl ether, p-methoxybenzyl ether, triethyl ether, alkyl ethers, and the like. aryl ethers; alkoxymethyl ethers such as methoxymethyl ether, 2-methoxyethoxymethyl, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, 2-(trimethylsilyl)ethoxymethyl ether, and the like; tetrahydropyranyl ether and related ethers; methylthiomethyl ether; esters such as acetate, benzoate, pivalate, methoxyacetate, chloroacetate, levulinate, and the like; and carbonates such as benzyl carbonate, p-nitrobenzyl carbonate, tert-butyl carbonate, 2,2,2-trichloroethyl carbonate, and 2-(trimethylsilyl)ethylallyl carbonate. Specific examples of the protecting group include alkyl ethers, such as methyl ether. X 2is a bridging group that allows attachment to polyglutamic acid groups and groups derived therefrom. In some cases, X 2 can be a group that allows for fixation with other PGAs and their derivatives.

[0077] Polymer-PGA / PEG In some cases, PEG is attached to PGA and its derivatives via the amine groups and / or carboxylic acids of PGA and its derivatives. PEGylation of the polymer can be carried out using any suitable method available in the art.

[0078] Such polymers can be used in a variety of molecular weights, for example, suitable PEG or PGA-PEG molecular weights are about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, about 10 kDa to about 50 kDa, or about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, and about 35 kDa.

[0079] As another example, the molecular weight of suitable PEG or PGA-PEG and its water-soluble derivatives can be about 1 kDa to about 50 kDa, about 2 kDa to about 40 kDa, about 3 kDa to about 30 kDa, or about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, or about 30 kDa.

[0080] As other non-limiting examples, the PEG or PGA-PEG has a molecular weight of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. In some cases, the PEG or PGA-PEG has a molecular weight of 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 10 kDa or less, 5 kDa or less, 3 kDa or less, or 1 kDa or less. Any combination of these is also possible, and for example, the molecular weight of PEG or PGA-PEG is about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, or about 10 kDa to about 50 kDa.

[0081] In some cases, PGA-PEG polymers and their water-soluble derivatives can be used with various degrees of PEGylation, where the degree of PEGylation is defined as the percentage of functional groups in the PGA or functional PGA derivatives functionalized with PEG. Thus, suitable degrees of PEGylated PGA-PEG polymers and their water-soluble derivatives can be, for example, about 0.1% to about 10%, about 0.2% to about 5%, about 0.5% to about 2%, or about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0082] In some embodiments, the percentage of PEG in the PGA-PEG and its derivative water-soluble polymers can be about 10% to 90% (w / w), about 15% to 80%, about 20% to 70%, or about 20%, about 22%, about 24%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, or about 60% by weight of the total weight of the polymer.

[0083] In some embodiments, the polymer comprises a water-soluble derivative of PGA or PGA-PEG, where the PGA is optionally substituted with one or more groups at one or more available positions, e.g., amine and / or carboxylic acid groups. Suitable derivatives of PGA and PGA-PEG derivatives include poly(alkylglutamines) and derivative PEG-poly(alkylglutamines), such as poly(N-2-(2'-hydroxyethoxy)ethyl-L-glutamine) (PEEG), PEG-PEEG, poly(N-3-(hydroxypropyl)-L-glutamine) (PHPG), PEG-PHPG, poly(N-2-(hydroxyethyl)-L-glutamine) (PHEG), PEG-PHEG, poly(alpha-benzyl-L-glutamate) (PBG), PEG-PBG, poly(ga Poly(aminoethyl-L-glutamine) (pPyAEG), PEG-pPyAEG, poly(aminoethyl-L-glutamine) (PAEG), PEG-PAEG, poly(histamino-L-glutamine) (pHisG), PEG-pHisG, poly(agmatine-L-glutamine) (pAgmG), and PEG-pAgmG, and mixtures thereof.

[0084] Polymer - Poly(aspartic acid), PASP In another set of embodiments, the polymer is a polymer of the amino acid aspartic acid, e.g., (PAsp) nThe polymer may include poly(aspartic acid) (PASP), which is:

[0023] . Any number of aspartic acid units may be present in the polymer. For example, n may be at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n may be 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Any combination of these is also possible, e.g., n is 2-100. It should also be noted that other amino acids may be present in the PASP chain, and the polymer may be linear or branched. Furthermore, as used herein, PASP includes water-soluble salts and water-soluble PASP and / or PASP derivatives.

[0085] The poly(aspartic acid) can have any suitable molecular weight, for example, the poly(aspartic acid) can have a molecular weight of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. In some cases, the poly(aspartic acid) has a molecular weight of 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 10 kDa or less, 5 kDa or less, 3 kDa or less, or 1 kDa or less, or any combination thereof, such as a poly(aspartic acid) having a molecular weight of about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, or about 10 kDa to about 50 kDa.

[0086] Furthermore, in some cases, the poly(aspartic acid) is PEGylated, for example, with one or more PEG moieties. The PEG has any of the formulas described herein. For example, the PEG is modified to allow the formation of a conjugate PASP-PEG. The modified PEG can be, for example, the following: X 1 -(O-CH2-CH2) n -X 2 wherein X 1 is a hydrogen or hydroxyl protecting group that blocks the OH radical functionality for subsequent reaction. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Combinations of any of these are also possible, e.g., n is 2-100.

[0087] Polymers with attached hydrophobic moieties In some embodiments, the polymer (e.g., PSA) is conjugated to a hydrophobic moiety. In some cases, the nanoentity is a micelle. In some cases, the nanoentity has an outer hydrophilic surface and a hydrophobic interior. The hydrophobic moiety comprises an alkyl group, e.g., a straight-chain alkyl group. In some embodiments, the hydrophobic moiety comprises at least two carbon atoms. In other embodiments, the hydrophobic moiety comprises at least three carbon atoms. In some embodiments, the hydrophobic moiety is a C2-C 24 Straight chain alkyl groups (e.g., C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C19 , C 20 , C 21 , C 22 , C 23 , and / or C 24 In certain embodiments, the hydrophobic moiety comprises a linear C 12 In some embodiments, the compositions of the present invention further comprise an aliphatic carbon chain covalently bonded to the polymer (e.g., PSA). In some embodiments, the aliphatic carbon chain is a C2-C 24 Aliphatic carbon chains (e.g. C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , and / or C 24 ) is included.

[0088] Non-limiting examples of hydrophobic moieties include C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24or other alkyl groups (e.g., straight or branched chain alkyl groups, e.g., isoalkyl groups). In some cases, the hydrophobic moiety comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 carbon atoms. The hydrophobic moiety is saturated or unsaturated, for example, containing one or more carbon-carbon double or triple bonds. One technique for attaching the hydrophobic moiety is, as a non-limiting example, by using C 12 In some cases, a hydrophobic moiety (e.g., C 12 Hydrophobic moieties are attached using activation with quaternary ammonium salts (e.g., tetrabutylammonium hydroxide) and tetrafluoroboric acid (e.g., 2-bromo-1-ethylpyridinium tetrafluoroborate) prior to reaction with an alkylamine such as dodecylamine of the formula (I). In other embodiments, other methods of attaching hydrophobic moieties are used, for example, using click chemistry, Grignard reaction, etc.

[0089] Other non-limiting examples of attached hydrophobic moieties include cycloalkanes (e.g., cyclopropane, cyclobutane, cyclopentane, cylcohexane, etc.), bile salts, terpenoids, terpenes, terpene-derived moieties, and fat-soluble vitamins, such as vitamins A, D, E, K, etc., and derivatives thereof. Non-limiting examples of bile salts include underivatized bile salts, such as cholate, deoxycholate, chenodeoxycholate, and urosodeoxycholate. Non-limiting examples of derivatized bile salts include taurocholate, taurodeoxycholate, tauroursodeoxycholate, taurochenodeoxycholate, glycolate, glycodeoxycholate, glycoursodeoxycholate, glycochenodeoxycholate, taurolithocholate, and glycolithocholate.

[0090] In another set of embodiments, at least a portion of the sialic acid or other monomers of the polymer are conjugated to polyethylene glycol (PEG), although it should be understood that PEG is not required in all embodiments. Polyethylene glycol (PEG), in its most common form, has the following formula: H-(O-CH2-CH2) p -OH where p is an integer representing the degree of PEG polymerization. In some cases, the PEG is also modified, for example: X 3 -(O-CH2-CH2)pX 4 wherein X 3is a hydrogen or hydroxyl protecting group that blocks the OH functionality for subsequent reaction. Protecting groups for hydroxyl radicals are well known in the art, and representative protecting groups (already containing the protecting oxygen) include, but are not limited to, silyl ethers, such as trimethylsilyl ether, triethylsilyl ether, tertbutyldimethylsilyl ether, tert-butyldiphenylsilyl ether, triisopropylsilyl ether, diethylisopropylsilyl ether, tetradimethylsilyl ether, triphenylsilyl ether, di-tert-butylmethylsilyl ether, etc., alkyl ethers, such as methyl ether, tert-butyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trityl ether, allyl ethers, etc.; alkoxymethyl ethers such as methoxymethyl ether, 2-methoxyethoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, 2-(trimethylsilyl)ethoxymethyl ether, etc., tetrahydropyranyl ether and related ethers; methylthiomethyl ether, esters such as acetates, benzoates, pivalates, methoxyacetates, chloroacetates, levulinates, carbonates such as benzyl carbonate, p-nitrobenzyl carbonate, tert-butyl carbonate, 2,2,2-trichloroethyl carbonate, 2-(trimethylsilyl)ethyl, allyl carbonate, etc. In one embodiment, the protecting group is an alkyl ether, such as a methyl ether.

[0091] X 4 represents the anchoring of the polymer to sialic acid or another monomer and is a covalent bond or cross-linking moiety, such as N-hydroxy-succinimide (NHS), a maleimide group, biotin, etc., which can bind to an amine, e.g., a primary amine, a sulfhydryl moiety, or avidin or streptavidin, respectively, on the modified sialic acid or other monomer. In some cases, X 3 is also a group that allows for attachment to, for example, sialic acid or another monomer.3 includes hydrophobically modified PSA or other polymers as discussed herein. For example, in one set of embodiments, hydrophobic moieties, such as C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , or another alkyl group (e.g., a straight or branched chain alkyl group, e.g., an isoalkyl group). In some cases, the hydrophobic moiety contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 carbon atoms. In some embodiments, the hydrophobic moiety is sufficiently hydrophobic that X 4 Compared to unmodified PSA without the X group, 4 PSA with X is more hydrophobic, e.g., 4 In an octanol / water partitioning system, the compound partitions more into octanol than when it does not have the group.

[0092] In one set of embodiments, PEG is attached to the polymer via an amine group and / or a carboxylic acid group. PEGylation can be carried out using any suitable method available in the art. See, for example, Gonzalez and Vaillard, "Evolution of Reactive mPEG Polymers for the Conjugation of Peptides and Proteins," Curr. Org. Chem., 17(9):975-998, 2013, and Giorgi, et al., "Carbohydrate PEGylation, an approach to improve pharmacological potency," Beilstein J. Org. Chem., 10:1433-44, 2014. PEG can be used in a variety of molecular weights, and the appropriate molecular weight for a given use can be easily determined by one of skill in the art. Thus, for example, suitable molecular weights of PEG are from about 1 kDa to about 100 kDa, from about 5 kDa to about 80 kDa, or from about 10 kDa to about 50 kDa, for example, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, or about 35 kDa.

[0093] In some embodiments, the degree of PEGylation is defined as the percentage of functional groups in the polymer that are functionalized with PEG. Examples of suitable degrees of PEGylation include about 0.1% to about 10%, about 0.2% to about 5%, or about 0.5% to about 2%, such as about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0094] In some embodiments, the percentage of PEG in the final polymer can be about 10% to 90% (w / w), about 15% to 80%, about 20% to 70%, or about 20% to 60% by weight of the total polymer, e.g., about 22%, about 24%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, or about 60%.

[0095] targeting part In one aspect, the entity also includes a targeting moiety, although it should be noted that in some embodiments, a targeting moiety is not present. The targeting moiety (if present) is used to target delivery of the entity, for example, to a specific cell population within a subject. For example, the targeting moiety facilitates access of the nanoentity to one type of cell, such as a cancer cell, endothelial cell, or immune cell. In some embodiments, the targeting moiety enables targeting of the entity to a specific location within a subject, such as a specific organ or a specific cell type (e.g., a tumor or cancer cell). In some cases, the entity is taken up by cells without the need for a targeting moiety, and in some other cases, the targeting moiety (e.g., the targeting moiety is a cell-penetrating peptide and / or a tissue-penetrating peptide, such as Lyp-1 or tLyp-1, or a CendR peptide or other peptide discussed herein) facilitates internalization. However, it should be understood that in certain embodiments, the targeting moiety may not necessarily also facilitate internalization. In some embodiments, more than one type of targeting moiety is present. In some embodiments, the targeting moiety comprises a cell-penetrating peptide and / or a tumor / tissue-penetrating peptide.

[0096] The subject can be a human or a non-human animal. Examples of subjects include, but are not limited to, mammals, such as cows, sheep, goats, horses, rabbits, pigs, mice, rats, dogs, cats, primates (e.g., monkeys, chimpanzees, etc.). In some cases, the subject is a non-mammal, such as a bird, an amphibian, or a fish.

[0097] A wide variety of targeting moieties may be used in various embodiments. For example, targeting moieties include peptides, proteins, aptamers, antibodies (including monoclonal antibodies, nanobodies, and antibody fragments), nucleic acids, organic molecules, ligands, etc. Non-limiting examples include insulin or transferrin.

[0098] For example, in one set of embodiments, the targeting moiety is a peptide, e.g., 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, or 10 amino acids or less in length. In certain embodiments, the targeting moiety comprises a cell recognition sequence, such as a sequence comprising RGD (arginine-glycine-aspartic acid). In certain embodiments, the targeting moiety comprises a cell recognition sequence, such as a sequence comprising NGR (asparagine-glycine-arginine).

[0099] "Amino acid" has its ordinary meaning as used in the field of biochemistry. Isolated amino acids may, but do not necessarily (e.g., as in the case of proline), have the general structure: [ka] In this structure, alpha (α) can be any suitable moiety; for example, alpha (α) can be a hydrogen atom, a methyl group, or an isopropyl group. A series of isolated amino acids can be linked to form a peptide or protein by reacting the -NH2 of one amino acid with the -COOH of another amino acid to form a peptide bond (-CO-NH-). In such cases, the R groups on the peptide or protein may each be referred to as an amino acid residue. As used herein, "naturally occurring amino acids" refers to the 20 amino acids commonly found in nature, usually in their L-isomer form, namely, alanine ("Ala" or "A"), arginine ("Arg" or "R"), asparagine ("Asn" or "N"), aspartic acid ("Asp" or "D"), cysteine ​​("Cys" or "C"), glutamine ("Gln" or "Q"), glutamic acid ("Glu" or "E"), glycine ("Gly" or "G"), histidine ("His" or "H"), and arginine ("Arg" or "R"), asparagine ("Asn" or "N"), aspartic acid ("Asp" or "D"), cysteine ​​("Cys" or "C"), glutamine ("Gln" or "Q"), glutamic acid ("Glu" or "E"), glycine ("Gly" or "G"), and histidine ("His" or "H"). ), isoleucine ("Ile" or "I"), leucine ("Leu" or "L"), lysine ("Lys" or "K"), methionine ("Met" or "M"), phenylalaine ("Phe" or "F"), proline ("Pro" or "P"), serine ("Ser" or "S"), threonine ("Thr" or "T"), tryptophan ("Trp" or "W"), tyrosine ("Tyr" or "Y"), and valine ("Val" or "V").

[0100] In one series of embodiments, the targeting moiety is a cell-penetrating peptide and / or tissue-penetrating peptide. Various cell-penetrating peptides can be used. For example, the peptide comprises a C-terminal "C-end Rule" (CendR) sequence motif (R / K)XX(R / K). The cell-penetrating peptide has the ability to penetrate cell membranes. In some cases, the cell-penetrating peptide and / or tissue-penetrating peptide also facilitates targeting of the nano-entity to cells. Each X in this sequence is independently an amino acid or a non-amino acid.

[0101] In some cases, the targeting moiety is 1 X 1 X 2 Z 2 Contains Z 1 is R or K, and Z 2 is R or K, and X 1 and X 2 are each independently an amino acid residue or a non-amino acid residue. In some cases, one or both termini of the peptide may have, for example, the structure J 1 Z 1 X 1 X 2 Z 2 , Z 1 X 1 X 2 Z 2 J 2 , or J 1 Z 1 X 1 X 2 Z 2 J 2 It contains other amino acids such as 1 and J. 2 are each independently an amino acid sequence (e.g., containing 1, 2, 3, 4, 5, 6, or more amino acid residues) or an aliphatic carbon chain. The aliphatic carbon chain may contain carbon and hydrogen atoms in any suitable arrangement, e.g., linear or branched, and may be saturated or unsaturated. For example, in one set of embodiments, the aliphatic carbon chain may have the formula, e.g., —(CH)—, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or any other positive integer. n -, and a straight chain alkyl. In addition, in some cases, the sequence may be, for example, CJ 1 Z 1 X 1 X 2 Z 2 , CZ 1 X 1 X 2 Z 2 J 2 , or C.J. 1 Z 1 X 1 X 2 Z 2 J 2As such, it ends with a cysteine ​​residue.

[0102] Non-limiting examples of CendR peptides include Lyp-1, tLyp-1, iNGR, cLyp1, iRGD, RPARPAR, TT1, or linear TT1. Optionally, other amino acids are present in the peptide. Lyp-1 has the sequence CGNKRTRGC (SEQ ID NO: 1). In some embodiments, two Cys residues are linked to each other via a disulfide bridge, thereby forming a cyclic structure. In some cases, only a portion of the Lyp-1 sequence is present, such as in the case of tLyp-1(CGNKRTR) (SEQ ID NO: 2). cLyp1 has the sequence CGNKRTRGC (SEQ ID NO: 3), with two cysteines linked to each other. iNGR has the sequence CRNGRGPDC (SEQ ID NO: 4), with two cysteines linked to each other. iRGD has the sequence (CRGDKGPDC) (SEQ ID NO: 5) or the sequence CRGDRGPDC (SEQ ID NO: 6), with two cysteines linked to each other. RPARPAR has the sequence RPARPAR (SEQ ID NO: 7). TT1 has the sequence CKRGARSTC (SEQ ID NO: 8), with two cysteines linked together. Linear TT1 has the sequence AKRGARSTA (SEQ ID NO: 9).

[0103] In some embodiments, the targeting moiety comprises the sequence RGD. Optionally, other amino acids may be present in the peptide. Non-limiting examples of RGD peptides include RGD, RGD-4C, cRGD, or cilengitide. Optionally, other amino acids may be present in the peptide. RGD has the sequence RGD (SEQ ID NO: 10). RGD-4C has the sequence CDCRGDCFC (SEQ ID NO: 11). cRGD has the sequence cRGDf(NMeV) (SEQ ID NO: 12) or c(RGDyK) (SEQ ID NO: 13). Cilengitide has the sequence cyclic-(N-Me-VRGDf-NH) (SEQ ID NO: 14).

[0104] In some embodiments, the targeting moiety comprises the sequence NGR. Optionally, other amino acids may also be present in the peptide.

[0105] For example, several targeting moieties can be found in Bertrand N., et al., Cancer Nanotechnology: The impact of passive and active targeting in the era of modern cancer biology, Advanced Drug Delivery Reviews 66 (2014) 2-25; Gilad Y., et al., Recent innovations in peptide-based targeted delivery to cancer cells, Biomedicines, 4 (2016); and Zhou G., et al. Aptamers: A promising chemical antibody for cancer therapy, Oncotarget, 7 (2016) 13446-13463. The targeting moiety can be a peptide, e.g., a CendR peptide (e.g., Lyp1, cLyp1, tLyp1, iRGD, iNGR, TT1, linear TT1, RPARPA, F3, etc.), an RGD peptide (e.g., 9-RGD, RGD4C, delta24-RGD, delta24-RGD4C, RGD-K5, cilengitide, acyclic RGD4C, bicyclic RGD4C, c(RGDfK), c(RGDyK), E-[c(RGDfK)2], E[c(RGDyK)]2). , NGR peptide, KLWVLPKGGGC (SEQ ID NO: 15), CDCRGDCFC (SEQ ID NO: 16), LABL, angiopeptin-2; proteins, such as transferrin, ankyrin repeat proteins, affibodies; small molecules, such as folic acid, triphenylphosphonium, ACUPA, PSMA, carbohydrate moieties (e.g., mannose, glucose, galactose, and derivatives thereof); and aptamers.

[0106] Peptides, including any of the sequences disclosed above, in some embodiments exhibit cell- or tissue-penetrating activity, particularly in tumor tissue. One set of embodiments generally relates to combining cell-penetrating peptides with non-targeting properties to, for example, confer cell- or tissue-penetrating activity on at least a portion of a nano-entity when administered non-systemically to a subject (e.g., intratumoral, nasal, topical, intraperitoneal, vaginal, rectal, oral, pulmonary, intraocular, etc.) or when administered, for example, to a living cell or tissue, in vitro or ex vivo. In some cases, a portion of a polymer (e.g., PSA) is attached to the cell-penetrating peptide, for example, by a non-covalent bond.

[0107] For example, several cell-penetrating peptides can be found in Zhang D. et al., Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems, Journal of Controlled Release, Volume 229 (2016) Pages 130-139, and Regberg J., et al. Applications of cell-penetrating peptides for tumor targeting and future cancer therapies, Pharmaceuticals, 5 (2012) 991-1007. Cell-penetrating peptides useful in certain embodiments of the present invention include TAT, mTAT (C-5H-TAT-5H-C), G3R6TAT, TAT(49-57), TAT(48-60), MPS, VP22, Antp, gH625, arginine-rich CPPs (e.g., octaarginine, polyarginine, stearyl-polyarginine, HIV-1 Rev34-50, FHV coat35-49) penetratin, penetratin-Arg, penetratin-Lys, SR9, HR9, PR9, H(7)K(R(2)), Pep-1, Pep-3, transportan, transportan 10, pepFect, pVEC, JB577, TD-1, MPG8, CADY, YTA2, YTA4, SynB1, SynB3, PTD-4, GALA, SPACE, and the like, but are not limited to these. The cell-penetrating peptide conjugated to the targeting moiety is selected from, but is not limited to, PEGA (CPGPEGAGC) (SEQ ID NO: 18), CREKA (SEQ ID NO: 19), RVG (YTIWMPENPRPGTPCDIFTNSRGKRASNG) (SEQ ID NO: 20), DV3 (LGASWHRPDKG) (SEQ ID NO: 21), DEVDG (SEQ ID NO: 22), ACPP-MMP-2 / 9 (PLGLAG) (SEQ ID NO: 23), ACPP-MMP-2 (IAGEDGDEFG) (SEQ ID NO: 24), R8-GRGD (SEQ ID NO: 25), penetratin-RGD, and the like.

[0108] For example, some tumor / tissue-penetrating peptides can be found in Ruoslahti E., Tumor penetrating peptides for improved drug delivery, Advanced Drug Delivery Reviews, Volumes 110-111 (2017) Pages 3-12. Tumor / tissue-penetrating peptides useful in certain embodiments of the present invention include CendR peptides, such as iRGD (C RGDK GPDC) (SEQ ID NO: 26), Lyp-1 (CGN KRTR GC) (SEQ ID NO: 27), tLyp-1 (CGN KRTR ) (SEQ ID NO: 28), TT1 (CK RGAR STC) (SEQ ID NO: 29), linear TT1 (AK RGAR STA) (SEQ ID NO: 30), iNGR (C RNGR GPDC) (SEQ ID NO: 31), RPARPAR, F3(KDEPQR RSAR LSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 32), and the like. In one embodiment, the tumor / tissue-penetrating peptide comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 22. In a further embodiment, the tumor / tissue-penetrating peptide consists of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 22.

[0109] In some cases, an antibody (including a nanobody, antibody fragment, monoclonal antibody, or other antibody) is attached to the surface or shell of an entity, such as a nanocapsule or other entity described herein.

[0110] Bond between the polymer and the targeting moiety In certain aspects, a portion of the polymer (e.g., PSA) is attached to the targeting moiety, e.g., by a covalent bond. The polymer is attached to the targeting moiety directly or indirectly, e.g., via a linker, e.g., an aminoalkyl(C1-C4) succinimide linker (including C1, C2, C3, and C4) or an aminoalkyl(C1-C4) amido-isopropyl linker (including C1, C2, C3, and C4). In some cases, other aminoalkyl succinimide or aminoalkyl amido-isopropyl linkers are used. In some embodiments, the targeting moiety includes a C-terminus for conjugation, e.g., in some cases. The aminoalkyl(C1-C4) succinimide linker is an aminoethyl succinimide linker, aminopropyl succinimide, aminobutyl succinimide, or the like. Aminoalkyl(C1-C4) succinimide linkers can be formed, for example, by attaching a maleimide moiety to a carboxylic acid moiety on a monomer unit (e.g., a sialic acid unit) using an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) coupling reaction. In some cases, an N-aminoalkyl(C1-C4) maleimide moiety, such as an N-aminoethylmaleimide moiety, is reacted with a carboxylic acid moiety on a monomer unit to form an amide bond, thereby linking the maleimide moiety to a polymer (e.g., PSA). Aminoalkyl(C1-C4) amido-isopropyl linkers can be formed, for example, using aminoethylmethacrylamide or N-(3-aminopropyl)methacrylamide in the presence of BOP / TBA (benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate / tetra-n-butylammonium hydroxide).The maleimide or methacryloyl moiety can then be reacted with a cysteine, thiol group, or other sulfur-containing moiety in the peptide, e.g., by Michael-type addition, to conjugate the peptide to a polymer (e.g., PSA) via an aminoalkyl(C1-C4) succinimide, such as an aminoethylsuccinimide linker (see, e.g., Figure 1), or via an aminoalkyl(C1-C4) amido-isopropyl linker.

[0111] In some embodiments, the polymer (e.g., PSA) is directly linked to the targeting moiety via an amide group. See, for example, Mojarradi, "Coupling of substances containing a primary amine to hyaluronan via carbodiimide-mediated amidation," Master's Thesis, Uppsala University, March 2011. The amide group can be generated, for example, by reacting a carboxylic acid moiety on a monomer unit (e.g., a sialic acid unit) with a lysine, arginine, or other primary amine-containing moiety in the peptide; specifically, the primary amine group is located on the lysine or arginine amino acid moiety on the targeting moiety. In some embodiments, an activating agent is present in the reaction to form an intermediate, such as carbodiimide, N-hydroxysuccinimide, or DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) (Carbohydrate Polymers, 108, (2014), 239-246).

[0112] Additionally, in certain embodiments of the present invention, the entity comprises a penetration enhancer capable of facilitating cellular internalization or tissue penetration.

[0113] Thus, one set of embodiments generally relates to methods of reacting carboxylate moieties on a polymer (e.g., PSA) with aminoalkyl(C1-C4)maleimides and / or aminoalkyl(C1-C4)methacrylamides, and reacting the resulting aminoalkyl(C1-C4)maleimides and / or aminoalkyl(C1-C4)methacrylamides with cysteine ​​groups on a peptide to yield polymer-aminoalkyl(C1-C4)succinimide-peptide and / or polymer-aminoalkyl(C1-C4)amide isopropyl-peptide compositions.

[0114] Another set of embodiments relates to methods of reacting carboxylate moieties on a polymer (e.g., PSA) with N-hydroxysuccinimide or carbodiimide and reacting the formed intermediate with lysine or arginine groups on a peptide to yield a polymer-amide-peptide.

[0115] Medicines / drugs In various embodiments, the nanoentity comprises any of a variety of pharmaceuticals or drugs, which may be located within and / or on the surface of the nanoentity depending on the embodiment. One, two, three, or more pharmaceuticals or drugs may be present, for example, within the interior portion of the nanoentity. For example, the pharmaceuticals or drugs have a size or molecular weight that allows them to be contained within the interior portion of the nanoentity. For example, the pharmaceuticals or drugs are small molecules, e.g., with a molecular weight of less than 2000 Da. In some cases, the small molecules have a molecular weight of less than 1000 Da. In some embodiments, the molecular weight is less than 500 Da or 200 Da.

[0116] In some cases, a pharmaceutical product includes any substance or mixture of substances intended for use in the manufacture of a formulation, which, when used in the manufacture of a drug, is an active ingredient in the formulation, that provides pharmacological activity and / or other direct effects in the diagnosis, cure, mitigation, treatment, or prevention of disease, or that affect the structure and function of the body.

[0117] Examples of pharmaceutical agents include any pharmaceutically active chemical or biological compound that produces a pharmacological effect and is used to treat or prevent a disease state, any pharmaceutically acceptable salt thereof, and any mixture thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, nitrate, phosphate, hydrobromide, maleate, malate, ascorbate, citrate, tartrate, pamoate, laurate, stearate, palmitate, oleate, myristate, lauryl sulfate, naphthalenesulfonate, linoleate, linolenate, and the like. In some cases, the pharmaceutically acceptable salt is a sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, ammonium salt, and the like.

[0118] Pharmaceuticals or drugs are considered to be lipophilic, water-soluble, or amphiphilic (containing both non-polar and polar groups simultaneously and tending to form micelles in aqueous media). Given the complexity of classifying pharmaceuticals or drugs based solely on their solubility, for the sake of simplicity, and in no way limiting, reference will be made to the following two classes of drugs: lipophilic (compounds that exhibit some solubility in media containing oils and / or lipids and / or organic solvents, with a log P greater than 1.5) and water-soluble (compounds that exhibit some solubility in aqueous media, with a log P less than 1.5), where log P is defined as the octanol-water partition coefficient.

[0119] In certain embodiments, the pharmaceutical or drug is lipid-soluble, e.g., it can be contained within the non-aqueous interior of a nanocapsule or other entity, e.g., in an oil, lipid, and / or organic solvent, e.g., an organic solvent mixed with an oil. Furthermore, in some cases, the lipid-soluble pharmaceutical or drug is present on the outer surface or shell of the entity. Non-limiting examples of organic solvents include, but are not limited to, ethanol, butanol, 2-ethylhexanol, isobutanol, isopropanol, methanol, propanol, propylene glycol, acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl isopropyl ketone, mesityl oxide, trichloroethylene, ethylene bromide, chloroform, ethylene chloride, dichloromethane, tetrachloroethylene, carbon tetrachloride, dimethylformamide, 1,4-dioxane, butyl ether, dimethylformamide ethyl ether, diisopropyl ether, tetrahydrofuran, tert-butyl methyl ether, dimethyl sulfoxide, pyridine, cyclohexane, hexane, acetonitrile, ethyl acetate, toluene, xylene, and combinations thereof, and / or other organic solvents. In some cases, lipid-soluble drugs are generally hydrophobic in nature, eg, having a log P greater than 1.5, where P is the intrinsic octanol-water partition coefficient.

[0120] Non-limiting examples of lipid-soluble pharmaceuticals or drugs that can be used include, but are not limited to, the following: chemotherapeutic or anti-cancer agents, such as taxoids (e.g., docetaxel, paclitaxel, cabazitaxel), tomudex, daunomycin, aclarubicin, bleomycin, dactinomycin, daunorubicin, rapamycin, epirubicin, valrubicin, idarubicin, mitomycin C, mitoxantrone, elesclomol, ingenol mebutate, plicamycin, calicheamicin, Esperamycin, degarelix, emtansine, maytansine, maytansinoids (e.g., maytansinoid DM1, maytansinoid 2, maytansinoid DM4), mitomycin, auristatins, vinorelbine, vinblastine, vincristine, vindesine, estramustine, hydrophobic derivatives of cisplatin, chlorambucil, bendamustine, carmustine, amantadine, rimantadine, lomustine, semustine, amsacrine, ladribine, cytarabine, (C 12 ~C 18)-gemcitabine, tegafur, trimetrexate, epothilones A-E (e.g., sagopilone, ixapebilone, patupilone), eribulin, camptothecin, aminoglutethimide, diaziquone, levamisole, methyl-GAG, mitotane, mitoxantrone, testolactone, michelanin B, bryostatin-1, halomon, didemnin (e.g., plitidepsin), trabectedin, lurbinectedin, vorinostat, romidepsin, irinotecan, bortezomib, erlotinib, getifinib, imatinib, vemurafenib, crizotinib, vismodegib, tretinoin, alitretinoin, bexarotene, etc.; or immunomodulatory / immunosuppressive agents, such as imiquimod, cyclosporine, tacrolimus, pimecrolimus, everolimus, sirolimus, tensirolimus, azathioprine, leflunomide, mycophenolate, etc.; or steroid drugs, such as enzalutamide, abiterone, exemestane, fulvestrant, 2-methoxyestradiol, formestane, atamestane, gymnesterol, methylprotodioscin, physalin B, physalin D, physalin F, withaferin A, ginsenosides, azasteroids, cinobufagin, bufalin, dienogest, etc.; or conjugates of steroids and cytotoxic drugs (e.g., nucleosides, paclitaxel, chlorambucil, and metal complexes), such as paclitaxel-estradiol.

[0121] Other illustrative, non-limiting examples of lipid-soluble biologically active molecules include: analgesics and anti-inflammatory agents (e.g., aloxiprine, auranofin, azapropazone, benorilate, diflunisal, etodolac, fenbufen, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, etc.); anthelmintics (e.g., albenda, azole, bephenium hydroxynaphthoate, campendazole, dichlorophen, ivermectin, mebendazole, oxamniquine, oxfendazole, oxanthrene embonate, praziquantel, pyranthrene embonate, thiabendazole, etc.); antidiabetic agents (e.g., acetohexamide, chlorpropamide, glibenclamide, gliclazide, glipizide, tolazamide, tolbutamide, etc.); antidepressants (e.g., amoxapine, maprotiline, mianserin, nortriptyline, trazodone, trimipramine, etc.); Antifungal agents (e.g., amphotericin, butoconazole nitrate, clotrimazole, econazole nitrate, fluconazole, flucytosine, griseofulvin, itraconazole, ketoconazole, miconazole, natamycin, nystatin, sulconazole nitrate, terbinafine, terconazole, tioconazole, undecenoic acid, etc.); antimalarials (e.g., amodiaquine, chloroquine, chlorproguanil, halofantrine, mefloquine, proguanil, pyrimethamine, quinine sulfate, etc.); antimigraine agents (e.g., dihydroethanol, Antiprotozoal drugs (e.g., benznidazole, clioquinol, decoquinate, diiodohydroxyquinoline, diloxanide furoate, dinitrumide, fluzolidone, metronidazole, nimorazole, nitrofurazone, ornidazole, tinidazole, etc.); Antithyroid drugs (e.g., carbimazole, propylthiouracil, etc.); Antiarrhythmic drugs (e.g., amiodarone, disopyramide, flecainide acetate, quinidine sulfate, etc.);Antibacterial agents (e.g., benethamine penicillin, cinoxacin, ciprofloxacin, clarithromycin, clofazimine, cloxacillin, demeclocycline, doxycycline, erythromycin, ethionamide, imipenem, nalidixic acid, nitrofurantoin, rifampicin, spiramycin, sulfabenzamide, sulfadoxine, sulfamerazine, sulfacetamide, sulfadiazine, sulfafurazole, sulfamethoxazole, sulfapyridine, tetracycline, trimethoprim, etc.) anticoagulants (e.g., dicoumarol, dipyridamole, nicoumarone, phenindione, etc.); anxiolytics, neuroleptics, sedatives, and hypnotics (e.g., alprazolam, amilobarbitone, barbitone, bentazepam, bromazepam, bromperidol, brotizolam, butobarbitone, carbromal, chlordiazepoxide, chlormethiazole, chlorpromazine, clobazam, clotiazepam, clozapine, diazepam, droperidol, etinamate, flunanisone, flumethic ... lunitrazepam, fluopromazine, flupentixol decanoate, fluphenazine decanoate, flurazepam, haloperidol, lorazepam, lormetazepam, medazepam, meprobamate, methaqualone, midazolam, nitrazepam, oxazepam, pentobarbitone, perphenazine pimozide, prochlorperazine, sulpiride, temazepam, thioridazine, triazolam, zopiclone, etc.); corticosteroids (e.g., beclomethasone, betamethasone, budesonide, cortisone acetate, desoxycortisone, etc.); methasone, dexamethasone, fludrocortisone acetate, flunisolide, flucortolone, fluticasone propionate, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, etc.); antigout drugs (e.g., allopurinol, probenecid, sulfinpyrazone, etc.); diuretics (e.g., acetazolamide, amiloride, bendrofluazide, bumetanide, chlorothiazide, chlorthalidone, ethacrynic acid, furosemide, metolazone, spironolactone, triamterene, etc.);Beta-blockers (e.g., acebutolol, alprenolol, atenolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, etc.); cardiac inotropes (e.g., amrinone, digitoxin, digoxin, enoximone, lanatoside C, medigoxin, etc.); antiparkinsonian agents (e.g., bromocriptine, lisuride, etc.); histamine receptor antagonists (e.g., acrivastine, astemizole, cinnarizine, cyclizine, cyproheptadine, dimenhydrinate, flunarizine, loratadine, meclozine, oxatomide, terfenadine, etc.); lipid-regulating agents (e.g., bezafibrate, clofibrate, fenofibrate, gemfibrozil, probucol, etc.); nitrates and other antianginal agents (e.g., amyl nitrate, glyceryl trinitrate, These include isosorbide dinitrate, isosorbide mononitrate, pentaerythritol tetranitrate, and the like; nutritional supplements (e.g., beta-carotene, vitamin A, vitamin B2, vitamin D, vitamin E, vitamin K, and the like); opioid analgesics (e.g., codeine, dextropropyoxyphene, diamorphine, dihydrocodeine, meptazinol, methadone, morphine, nalbuphine, pentazocine, and the like); sex hormones (e.g., clomiphene citrate, danazol, ethinyl estradiol, medroxyprogesterone acetate, mestranol, methyltestosterone, norethisterone, norgestrel, estradiol, conjugated estrogens, progesterone, stanozolol, stibestrol, testosterone, tibolone, and the like). Of course, in certain embodiments, mixtures of lipid-soluble drugs may be used if therapeutically effective.

[0122] However, in other embodiments, the pharmaceutical agent or drug is water-soluble, for example, it can be contained in the aqueous interior of the nanocapsule or attached to the surface of the nanocapsule. In some cases, the water-soluble drug exhibits some degree of solubility in aqueous media (e.g., log P is less than 1.5, where P is the intrinsic octanol-water partition coefficient). Examples include, but are not limited to, any pharmaceutically acceptable salt of the above-mentioned lipid-soluble drugs, such as docetaxel or docetaxel trihydrate; for example, salts include chloride, sulfate, bromide, mesylate, maleate, citrate, phosphate, hydrochloride; sodium, calcium, potassium, magnesium, meglumine, ammonium salt, etc. In various embodiments, any suitable pharmaceutical agent or drug that can be contained in a suitable solvent within the nanocapsule is used, as discussed herein.

[0123] Other examples of water-soluble pharmaceuticals or drugs that may be used include, but are not limited to, the following: chemotherapeutic agents (e.g., topotecan, teniposide, etoposide, pralatrexate, omacetaxine, doxorubicin, dacarbazine, procarbazine, hydroxydaunorubicin, hydroxyurea, 6-mercaptopurine, 6-thioguanine, floxuridine or 5-fluorodeoxyuridine, fludarabine, 5-fluorouracil, methotrexate, thiotepa, gemcitabine, pentostatin, mechlorethamine, pivobroman, riboflavin ... oman, cyclophosphamide, ifosfamide, busulfan, carboplatin, picoplatin, tetraplatin, satrapalin, platinum-DACH, ormaplatin, oxaplatin, melphalan, aminoglutethimide, etc.); antibacterial agents (e.g., triclosan, cetylpyridium chloride, domiphen bromide, quaternary ammonium salts, zinc compounds, sanguinarine, fluoride, alexidine, octonidine, EDTA, etc.); nonsteroidal anti-inflammatory and pain-relieving agents (e.g., aspirin, acetaminophen, etc.); minophen, ibuprofen, ketoprofen, diflunisal, fenoprofen calcium, flurbiprofen sodium, naproxen, tolmetin sodium, indomethacin, celecoxib, valdecoxib, parecoxib, rofecoxib, etc.); antitussives (e.g., benzonatate, caramiphen edisylate, menthol, dextromethorphan hydrobromide, chlophedianol hydrochloride, etc.); antihistamines (e.g., brompheniramine maleate, chlorpheniramine maleate, carbinoxamine maleate, crema lutein fumarate, dexchlorpheniramine maleate, diphenylhydramine hydrochloride, azatadine maleate, diphenhydramine citrate, diphenhydramine hydrochloride, diphenylpyraline hydrochloride, doxylamine succinate, promethazine hydrochloride, pyrilamine maleate, tripelennamine citrate, triprolidine hydrochloride, acrivastine, loratadine, desloratadine, brompheniramine, dexbropheniramine, fexofenadine, cetirizine, montelukast sodium, etc.);Expectorants (e.g., guaifenesin, ipecac, potassium iodide, terpine hydrate, etc.); analgesics-antipyretics (e.g., salicylates, phenylbutazone, indomethacin, phenacetin, etc.); antimigraine drugs (e.g., sumitriptan succinate, zolmitriptan, valproic acid, eletriptan hydrobromide, etc.); H2-antagonists and / or proton pump inhibitors (e.g., ranitidine, famotidine, omeprazole, etc.);

[0124] In some cases, the interior portion may comprise peptides, proteins, or nucleotides, many of which are hydrophilic in nature. Additionally, in some cases, water-soluble pharmaceutical agents or drugs are present on the outer surface or shell of the entity. The peptide, protein, or nucleotide may have any type of activity, such as anti-tumor, anti-angiogenic, immunomodulatory / immunosuppressive, antigenic, anti-inflammatory, anti-pain, anti-migraine, anti-obesity, anti-diabetic, antibacterial, wound healing, anthelmintic, anti-arrhythmic, antiviral activity, anticoagulant activity, antidepressant, anti-epileptic, anti-fungal, anti-gout, antihypertensive, antimalarial, antimuscarinic, anti-protozoal, antithyroid, anxiolytic, sedative, hypnotic, neuroleptic activity, beta-blocker activity, cardiac inotropic, cell adhesion inhibitory activity, corticosteroid activity, cytokine receptor activity modulating, diuretic, anti-Parkinsonian, histamine H receptor antagonist activity, keratolytic, lipid regulating, muscle relaxant activity, anti-anginal, nutritional, stimulant activity, anti-erectile dysfunction activity, etc.

[0125] Examples of peptides and proteins include, but are not limited to, IL-27 interleukin, interferons (e.g., interferon alpha II, interferon alfacon-1, interferon alpha-n3, interferon gamma), parasporin 2, endostatin fragments, macromomycin, actinoxanthin, histidine-rich glycoprotein, carboxypeptidase G2, pancreatic ribonuclease, mitomarcin, arginine deiminase, protein P-30 or onconase, metalloproteinase inhibitors. agents, guanylate kinase, beclin-1, alloferon, ribonuclease mitogillin, aurein, CD276 antigen, dermaseptin-B2, lactoferricin B, plantaricin A, maximin, cecropin, human neutrophil peptide, caerin, nisin, maculatin, mCRAMP, BMAP-27, BMAP-28, citropin, human insulin, recombinant insulin, insulin analogs (e.g., insulin lispro, insulin aspart, insulin glulisine, insulin detemir, insulin degludec, insulin glargine, NPH insulin, etc.), GLP-1 analogues (e.g., exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, taspoglutide, semaglutide, etc.), GLP-2 analogues (e.g., teduglutide), somatropin, anakinra, dornase alfa, whey acidic protein, SPARC or osteonectin protein, protein C, keratin subfamily A, human growth hormone or somatotropin, gonadotropins, angiopoietins, colony-stimulating factors (e.g., macrophage colony-stimulating factor, granulocyte colony-stimulating factor, stimulating factor, granulocyte-macrophage colony-stimulating factor, etc.), epidermal growth factor, erythropoietin, fibroblast growth factor, GDNF family of ligands, growth differentiation factor-9, hepatocyte growth factor, hepatocellular carcinoma-derived growth factor, insulin-like growth factor, keratinocyte growth factor, macrophage-stimulating protein, neurotrophins, placenta growth factor, platelet-derived growth factor, thrombopoietin, transforming growth factor, vascular endothelial growth factor, chemokines, interleukins, lymphokines, tumor necrosis factors (e.g., tumor necrosis factor alpha), Fc fusion proteins,These include contulakin G peptides and derivatives, antiphlamins, opioid peptides, lipopeptides (e.g., surotomycin), antigens such as tetanus and diphtheria toxoids, hepatitis B, and antibodies such as monoclonal antibodies (mAbs). Thus, by way of non-limiting example, nanoentities such as nanocapsules contain, for example, monoclonal antibodies or small molecules within the interior portion of the entity, the exterior portion, or both. Of course, in certain embodiments, mixtures of water-soluble drugs may be used where therapeutically effective.

[0126] As used herein, "antibody" refers to a protein or glycoprotein having one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which further define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kD) and one "heavy chain" (approximately 50-70 kD). The N-terminus of each chain defines a variable region, primarily responsible for antigen recognition, consisting of approximately 100-110 or more amino acids. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments obtained by digestion with various peptidases. Thus, for example, pepsin digestion of an antibody below the disulfide bond in the hinge region (i.e., toward the Fc domain) produces a Fab dimer, F(ab)'2, which itself is a light chain linked to VH-CH1 by a disulfide bond. This F(ab)'2 is reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. This Fab' monomer is essentially a Fab with a portion of the hinge region. While various antibody fragments are defined in terms of digestion of intact antibodies, these and other fragments can also be synthesized chemically de novo, for example, by using recombinant DNA technology, "phage display" methods, etc. Exemplary antibodies include single chain antibodies, such as single chain Fv (scFv) antibodies, in which a variable heavy chain and a variable light chain are linked (directly or via a peptide linker) to form a contiguous polypeptide.Other non-limiting examples of antibodies include nanobodies, antibody fragments, monoclonal antibodies, chimeric antibodies, reverse chimeric antibodies, etc. Antigen-binding fragments include Fab, Fab', F(ab)2, dsFv, sFv, unibodies, minibodies, diabodies, triabodies, tetrabodies, nanobodies, probodies, domain bodies, unibodies, bispecific single-chain variable fragments (bi-scFv), etc.

[0127] Exemplary antibodies include, but are not limited to, trastuzumab, bevazizumab, durvalumab, nivolumab, inotuzumab, avelumab, pembrolizumab, olaratumumab, atezolizumab, daratumumab, elotuzumab, necitumumab, dinutuximab, blinatumomab, ramucirumab, obinutuzumab, denosumab, ipilimumab, brentuximab, ofatumumab, and combinations thereof.

[0128] Examples of nucleotides include, but are not limited to, DNA, RNA, siRNA, mRNA, miRNA, PNA, etc. Nucleotides may be sense or antisense in various embodiments.

[0129] The pharmaceutical agent is present at up to about 50% by weight based on the total dry weight of the components of the system, although the appropriate percentage will depend on various factors, such as the pharmaceutical agent being incorporated, its indication for use, and administration efficiency. For example, in some cases, the pharmaceutical agent is present at up to about 10% by weight or up to about 5% by weight. In certain embodiments, there are two or more pharmaceutical agents present, which may be dissolved in the same solution or separately, depending on the nature of the active pharmaceutical ingredients being incorporated.

[0130] In some embodiments, the nanoentity comprises one or more surfactants. In some embodiments, the shell of the nanoentity comprises one or more surfactants. In other embodiments, the interior portion of the nanoentity comprises one or more surfactants. The surfactants, if present, comprise any of a variety of components having structures and / or functional groups that allow them to simultaneously interact with the lipophilic and hydrophilic portions of the formulation. Examples of surfactants include, but are not limited to, the following: polyoxyethylene sorbitan monooleate (Polysorbate 80; Tween 80®; HLB 15), polyoxyethylene sorbitan monostearate (Tween® 60, HLB 14.9 and Tween 61®; HLB 9.6), polyoxyethylene sorbitan monooleate (Tween 81®; HLB 10), polyoxyethylene sorbitan tristearate (Tween 65®; HLB 10.5), polyoxyethylene sorbitan trioleate (Tween 85®; HLB 11), polyoxyethylene sorbitan monolaurate (Tween® 20, HLB 16.7 and Tween 21®; HLB 13.3), polyoxyethylene sorbitan monopalmitate (Tween® 40, HLB 15.6); PEGylated fatty acid esters and mixtures with PEG, polyethylene glycol monostearate (HLB 11.6), polyethylene glycol stearate, polyethylene glycol 40 stearate (HLB 17), polyethylene glycol 100 stearate (HLB 18.8), polyethylene glycol 400 dilaurate (HLB 9.7), polyethylene glycol 200 dilaurate (HLB 5.9), polyethylene glycol monopalmitate (HLB 11.6), Kolliphor HS15® (HLB 15), polyethylene glycol-15-hydroxystearate (HLB 14-16), D-alpha-tocopheryl polyethylene glycol succinate (TPGS; HLB 13.2), triethanolammonium oleate (HLB 12), sodium oleate (HLB 18), sodium cholate (HLB 18), sodium deoxycholate (HLB 16), sodium laurate (HLB 40), sodium glycolate (HLB 16-18), triethanolamine oleate (HLB 12), gum tragacanth (HLB 11.9), and sodium dodecyl sulfate (HLB 40); poloxamer 124 (HLB 16), poloxamer 188 (HLB 29), poloxamer 237 (HLB 29), poloxamer 238 (HLB 28), poloxamer 278 (HLB 28), poloxamer 128 (HLB 16), poloxamer 189 (HLB 29), poloxamer 239 (HLB 28), poloxamer 278 (HLB 28), poloxamer 129 ... Poloxamer 338 (HLB 27) and Poloxamer 407 (HLB 22), sorbitan monooleate (Span® 80, HLB 4.3), sorbitan monolaurate (Span® 20, HLB 8.6), sorbitan monostearate (Span® 60, HLB 4.7), sorbitan trioleate (Span® 85, HLB 1.8), sorbitan sesquioleate (Span® 83, HLB 3.7), sorbitan monopalmitate (Span® 40, HLB 6.7), sorbitan isostearate (Span® 120, HLB 4.7), lauroyl macrogolglycerides (e.g., Gelucire® 44 / 14, HLB 14, and Labrafil® M2130CS, HLB 4), stearoyl macrogolglycerides (e.g., Gelucire® 50 / 13, HLB 13), linoleoyl macrogolglycerides (e.g., Labrafil® M2125CS, HLB 4), oleoyl macrogolglycerides (Labrafil® M1944CS, HLB 4), caprylocaproyl macrogolglycerides (Labrasol®, HLB 14), lecithin (e.g., egg yolk) Examples of surfactants include lecithin, soy lecithin, non-GMO lecithin, rapeseed lecithin, sunflower lecithin, lysolecithin, etc.), phospholipids (e.g., egg phospholipids, soy phospholipids, synthetic phospholipids, hydrogenated phospholipids, PEGylated phospholipids, phosphatidylcholine, lysophosphaditylcholine, phosphadidylethanolamine, phosphatidylserine, etc.), Phosal®, Phospholipon®, or any combination of any of these and / or other surfactants. In some cases, the surfactant is cationic, such as benzethonium chloride, benzalkonium chloride, CTAB (hexadecyltrimethylammonium bromide), cetrimide, tetradecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, etc. In some cases, the cationic surfactant comprises an ammonium salt, e.g., as a head group. For example, the head group may include a primary, secondary, tertiary, or quaternary ammonium salt. Furthermore, it should be understood that such surfactants may not be required in all embodiments.

[0131] In some embodiments, the entity comprises at least one cationic surfactant, such as those described above. For example, certain embodiments of the invention generally relating to nanocapsules may, in some cases, comprise a surfactant, such as a cationic surfactant. For example, certain embodiments of the invention generally relating to nanocapsules and having a targeting moiety may further comprise a cationic surfactant.

[0132] Method for making compositions of entities Various aspects of the present invention also relate generally to systems and methods for making compositions, such as those described herein, e.g., nanoparticles, nanocapsules, micelles, or other nano-entities. In some cases, the composition is a pharmaceutical composition.

[0133] As an example, in one set of embodiments, nanoentities, e.g., nanocapsules, are fabricated using a one-step solvent diffusion method. In some cases, this involves preparing an aqueous solution containing a polymer (e.g., PSA) and, optionally, one or more water-soluble surfactants, preparing an oily solution (e.g., containing an oil, one or more surfactants, an organic solvent, etc.), and mixing the solutions. In some cases, the organic solvent is completely or partially evaporated.

[0134] In another set of embodiments, a two-stage solvent diffusion method may be used. For example, in some cases, this method involves preparing an oily solution (e.g., containing oil, one or more surfactants, an organic solvent, etc.) and adding it to an aqueous phase (or adding the aqueous phase on top of the oily phase). The aqueous phase optionally contains one or more water-soluble surfactants. The solution is stirred to form a nanoemulsion. In some cases, the organic solvent is completely or partially evaporated. After the nanoemulsion is formed, an aqueous solution containing a polymer (e.g., PSA) is added under stirring to create nanocapsules.

[0135] In another series of embodiments, an ultrasonic treatment method is used. For example, in some cases, this method involves preparing an oily solution containing oil, one or more surfactants, and optionally an organic solvent, and adding it to an aqueous phase (or adding the aqueous phase on top of the oily phase). The aqueous phase optionally contains one or more water-soluble surfactants. The solution is mixed while being ultrasonicated to form a nanoemulsion. In some cases, the organic solvent is completely or partially evaporated. As already described in the solvent diffusion method, a polymer (e.g., PSA) is dissolved in the aqueous phase before ultrasonic treatment (one-step nanocapsule formation) or after obtaining a nanoemulsion by ultrasonic treatment (two-step process).

[0136] In another embodiment, the present invention relates to a method for encapsulating a pharmaceutical agent. In one embodiment, the pharmaceutical agent may be dissolved in an aqueous phase before preparing the nanoentities. In another embodiment, the pharmaceutical agent may be incubated with the nanoentities.

[0137] In another embodiment, the pharmaceutical agent is a monoclonal antibody, which is encapsulated by dissolving it in the aqueous phase prior to preparing the nanocapsules.

[0138] In another series of embodiments, a homogenization method is used. For example, in some cases, this method involves preparing an oily solution containing oil, one or more surfactants, and optionally an organic solvent, and adding it to an aqueous phase (or adding the aqueous phase on top of the oily phase). The aqueous phase optionally contains one or more water-soluble surfactants. The solution is mixed while homogenizing to form a nanoemulsion. In some cases, the organic solvent is completely or partially evaporated. As already described for both the solvent diffusion method and the ultrasonic treatment method, a polymer (e.g., PSA) is dissolved in the aqueous phase before homogenization (one-step nanocapsule formation) or after homogenization to obtain a nanoemulsion (two-step process).

[0139] In another embodiment, the emulsion is prepared using a self-emulsification method, for example, as discussed herein. For example, in some cases, this method involves preparing an oily solution containing oil and one or more surfactants (and optionally cosolvents), and adding it to an aqueous phase (or adding the aqueous phase to the oily phase). The aqueous phase optionally contains one or more water-soluble surfactants. In one series of embodiments, the emulsion is prepared without using a cosolvent (e.g., ethanol, PEG, glycerin, propylene glycol, etc.). As previously described, a polymer (e.g., PSA) is dissolved in the aqueous phase before self-emulsification (one-step nanocapsule formation) or after obtaining a nanoemulsion (two-step process).

[0140] In another embodiment, the present invention relates to a method for making nanoentities, the method comprising an additional freeze-drying step that can protect the nanoentities during storage. In some cases, it is not necessary to use a cryoprotectant during freeze-drying. In some embodiments, the nanoentities do not form aggregates upon reconstitution of the lyophilizate, so dilution of the colloidal system prior to freeze-drying is not necessary. In some cases, it is possible to add one or more sugars, e.g., sugars that exhibit cryoprotectant properties. Examples of cryoprotectants include, but are not limited to, trehalose, glucose, sucrose, mannitol, maltose, polyvinylpyrrolidone (PVP), glycerol, polyethylene glycol (PEG), propylene glycol, 2-methyl-2,4-pentanediol (MPD), raffinose, dextran, fructose, stachyose, and the like. In some cases, the cryoprotectant or other additives have other functions, e.g., as a buffer to control pH. In freeze-dried form, the nanoentities can be stored for extended periods and regenerated, e.g., by adding water.

[0141] Administration of the Composition Another aspect provides a method for administering any of the compositions discussed herein to an organism. When administering a composition of the present invention, it is administered in a therapeutically effective amount in a pharmaceutically acceptable formulation. As used herein, the term "pharmaceutically acceptable" means that the formulation contains agents or excipients that are compatible with the form required for administration to the organism and do not cause adverse effects. Any composition of the present invention is administered to an organism in a therapeutically effective amount. As used herein, "therapeutically effective" or "effective" refers to the amount necessary to delay the onset of, inhibit the progression of, completely halt the onset or progression of, diagnose, or otherwise achieve a medically desirable result for the specific condition being treated. The terms "treat," "treated," "treating," and the like generally refer to the administration of a composition of the present invention to an organism. When administered to an organism, the effective amount will depend on the specific condition being treated and the desired outcome. A therapeutically effective amount can be determined by one of ordinary skill in the art using no more than routine experimentation, using factors such as those described in detail below. For example, in one embodiment, the compositions herein are used to treat cancer by administering docetaxel to an organism, eg, intravenously.

[0142] Some embodiments of the present invention generally relate to the use of the compositions disclosed herein for the preparation of a medicament. For example, certain embodiments refer to the compositions disclosed herein for use in treating cancer.

[0143] When administering the compositions of the present invention to an organism, the dosage, schedule, route of administration, etc. are selected to affect the known activity of these compositions. Dosages are estimated based on the results of experimental models, optionally in combination with the results of assays for the compositions of the present invention. Dosages are adjusted appropriately depending on the mode of administration to achieve the desired local or systemic drug levels. Administration may be performed in one or more doses per day, week, or month.

[0144] Administration of a composition to an organism is intended to ensure that a therapeutically effective amount of the composition reaches the composition's site of action within the organism. In some cases, administration is performed at the maximum dose while avoiding or minimizing potential adverse side effects within the organism. The dose of the composition administered depends on factors such as the desired final concentration at the site of action, the method of administration to the organism, the effect of the composition, the tolerance of the composition within the organism, the timing of administration, and the impact of any current treatment. The dose delivered may also depend on conditions associated with the organism and, in some cases, may vary from organism to organism. For example, age, sex, weight, size, environment, physical condition, or the organism's current health status may affect the required dose and / or the concentration of the composition at the site of action. Dosage variations may occur between different individuals or even from day to day within the same individual. In some cases, a maximum dose is used, i.e., the highest safe dose according to sound medical judgment. In some cases, the dosage form should not substantially adversely affect the organism.

[0145] In certain embodiments, the composition of the present invention is administered to an organism with cancer.The administration of the composition of the present invention can be achieved by any medically acceptable method that allows the composition to reach its target.The specific mode selected will naturally depend on factors such as those previously described, such as the specific composition, the severity of the condition of the organism being treated, and the dosage required to achieve a therapeutic effect.As used herein, a "medically acceptable" treatment mode refers to a mode that delivers an effective level of the composition to the organism without causing clinically unacceptable adverse effects.

[0146] Any medically acceptable method can be used to administer the composition to an organism. Administration can be localized (i.e., confined to a specific region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated. For example, the composition can be administered orally or by other techniques, such as vaginally, rectally, bucally, pulmonary, topically, nasally, transdermally, intratumorally, by parenteral injection or implantation, surgically, or by any other method that allows the composition of the present invention to access its target. Compositions suitable for oral administration can be provided as discrete units, such as hard or soft capsules, pills, sachets, tablets, troches, or lozenges, each containing a predetermined amount of the active compound. Other oral compositions suitable for use with the present invention include solutions or suspensions in aqueous or non-aqueous liquids, such as syrups, elixirs, or emulsions. In another set of embodiments, the composition can be used to enhance the nutritional value of foods or beverages. In some embodiments, rectal administration may be used, for example, in the form of an enema, suppository, or foam.

[0147] In one set of embodiments, administration of the composition is parenteral, intratumoral, or oral. In some embodiments, the composition is administered by injection or infusion. In one embodiment, the injection is selected from intratumoral, subcutaneous, intramuscular, or intravenous injection. In another embodiment, the composition is administered by intrathecal injection or infusion.

[0148] In certain embodiments of the present invention, the administration of the composition of the present invention is designed to provide continuous exposure to the composition for a certain period of time, for example, for several hours, several days, several weeks, several months, or several years.This can be achieved, for example, by repeatedly administering the composition of the present invention by one of the above-mentioned methods.The administration of the composition can be alone or in combination with other therapeutic agents and / or compositions.

[0149] In certain embodiments of the present invention, the compositions may be combined with a suitable pharmaceutically acceptable carrier, for example, in dissolved or colloidal form, for example, incorporated into a polymeric release system or suspended in a liquid. Pharmaceutically acceptable carriers suitable for use in the present invention are generally well known to those skilled in the art. As used herein, "pharmaceutically acceptable carrier" refers to a non-toxic material used as a formulation component that does not significantly interfere with the effectiveness of the biological activity of the administered active compound(s), but that, for example, stabilizes or protects the active compound(s) in the composition prior to use. The term "carrier" refers to a natural or synthetic organic or inorganic component that, as discussed herein, combines with one or more active compounds of the present invention to facilitate application of the composition. Carriers are mixed or otherwise blended with one or more compositions of the present invention and with each other so as to avoid any interactions that would substantially impair the desired pharmaceutical effect. Carriers may be soluble or insoluble depending on the intended use. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble. Those skilled in the art will know of other suitable carriers, or will be able to ascertain such using no more than routine experimentation.

[0150] In some embodiments, the compositions of the present invention may include a pharmaceutically acceptable carrier along with formulation ingredients used with the active compound, such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, fillers, desiccants, antioxidants, antimicrobial agents, preservatives, binders, bulking agents, silica, solubilizers, or stabilizers, etc. For example, if the formulation is a liquid, the carrier may be a solvent, partial solvent, or non-solvent, and may be aqueous or organic. Examples of suitable formulation ingredients include diluents such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; lubricants such as magnesium stearate, stearic acid, or talc; retardants such as glycerol monostearate or glycerol distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, dispersing or wetting agents such as lecithin or other natural phospholipids; thickening agents such as cetyl alcohol or beeswax; buffers such as acetic acid and its salts, citric acid and its salts, boric acid and its salts, or phosphoric acid and its salts; or preservatives such as benzalkonium chloride, chlorobutanol, parabens, or thimerosal. The appropriate carrier concentration can be determined by one skilled in the art using no more than routine experimentation. The compositions contemplated herein can be formulated into solid, semi-solid, liquid, or gaseous form preparations, such as tablets, capsules, elixirs, powders, granules, ointments, solutions, deposits, inhalants, or injectables. Those of ordinary skill in the art will know of other suitable formulation ingredients, or will be able to ascertain such using no more than routine experimentation.

[0151] Preparations include, in certain embodiments, sterile aqueous or non-aqueous solutions, suspensions, and emulsions that can be isotonic with the blood of an organism. Examples of non-aqueous solvents include fixed oils, including polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, peanut oil, and mineral oil, injectable organic esters such as ethyl oleate, or synthetic monoglycerides or diglycerides. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present. Those of ordinary skill in the art can readily determine, without undue experimentation, the various parameters for preparing and formulating the compositions discussed herein.

[0152] The present invention also provides any of the compositions described above in the form of a kit, optionally including instructions for using the composition for the treatment of cancer or other diseases. Instructions may be provided for administering the composition by any suitable technique previously described, for example, orally or intravenously.

[0153] The composition of the present invention can be in the form of a kit.The kit generally defines a package containing any one or a combination of the composition of the present invention and other components described above.The kit can also include other containers containing one or more solvents, surfactants, preservatives, and / or diluents (e.g., normal saline (0.9% NaCl) or 5% dextrose), as well as containers for mixing, diluting, or administering the composition to an organism.

[0154] The composition of the kit can be provided as a liquid solution or dry powder. If the composition is provided as a dry powder, it can be reconstituted by adding a suitable solvent. In embodiments using a liquid composition, the liquid form can be concentrated or ready to use. The solvent depends on the composition and the mode of use or administration.

[0155] In the United States and other countries, where applicable, Spanish Patent Application No. P201731277, filed November 2, 2017, entitled "Sistemas de Liberacion de Farmacos de Acido Polisialico y Metodos," is hereby incorporated by reference in its entirety.

[0156] While multiple embodiments of the invention are described and illustrated herein, those skilled in the art will readily envision numerous other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily understand that any parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications employing the teachings of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the above-described embodiments are described by way of example only, and it should be understood that the invention may be practiced otherwise than as specifically described and claimed, within the scope of the appended claims and equivalents thereto. The present invention relates to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the scope of the present invention, unless such features, systems, articles, materials, kits, and / or methods are mutually exclusive.

[0157] If the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, the document having the later publication date shall control.

[0158] All definitions and definitions used herein are intended to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0159] The indefinite articles "a" and "an," as used in the specification and claims, unless otherwise specified, should be understood to mean "at least one."

[0160] The term "and / or," as used in the specification and claims, should be understood to mean "one or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, "A and / or B," in conjunction with open-ended language such as "comprising," may refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements), etc.

[0161] As used in this specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, i.e., including not only at least one element of a number or list of elements, but also two or more elements, and optionally, other unlisted items. Unless terms clearly state otherwise, such as "only one of" or "exactly one of," or "consisting of" as used in the claims, refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by exclusive terms such as "either," "one of," "only one of," or "exactly one of."

[0162] The phrase "at least one," as used herein and in the claims, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also recognizes that, optionally, elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically identified, may be present. Thus, as non-limiting examples, "at least one of A and B" (or, equivalently, "at least one of A or B," or "at least one of A and / or B") can refer to, in one embodiment, at least one A, optionally including more than one, and no B (and optionally including elements other than B); in another embodiment, at least one B, optionally including more than one, and no A (and optionally including elements other than A); in yet another embodiment, at least one A, optionally including more than one, and at least one B, optionally including more than one (and optionally including other elements); etc.

[0163] When the word "about" is used herein in reference to a number, it should be understood that yet another embodiment of the present invention includes that number unmodified by the presence of the word "about."

[0164] It should also be understood that for any method claimed herein that includes two or more steps or actions, unless expressly stated to the contrary, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

[0165] In the claims, as in the specification above, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," etc., are to be understood to be open-ended, i.e., meaning including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0166] Aspects / embodiments of the invention in so-called claim form: 1. (Embodiment 1): A composition comprising a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell comprising polysialic acid, and the inner portion comprising at least one hydrophobic compound.

[0167] 2. The composition of claim 1, wherein at least a portion of the plurality of nano-entities further comprise a targeting moiety and / or a cell-penetrating peptide and / or a tumor / tissue-penetrating peptide.

[0168] 3. The composition of claim 2, wherein the targeting moiety is electrostatically bound to the polysialic acid.

[0169] 4. The composition of claim 2, wherein the targeting moiety is attached to the polysialic acid via a linker.

[0170] 5. The composition of claim 2, wherein the targeting moiety is attached to the polysialic acid via an aminoalkyl(C1-C4)maleimide linker, an aminoalkyl(C1-C4)methacrylamide linker, or directly through the amide group.

[0171] 6. The composition of claim 5, wherein the aminoalkyl(C1-C4)maleimide linker is generated by an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) or DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) coupling reaction.

[0172] 7. The composition of claim 5, wherein the targeting moiety is attached to the polysialic acid via an aminoethylmaleimide linker.

[0173] 8. The composition of any one of claims 2 to 7, wherein the targeting moiety comprises a peptide or protein.

[0174] 9. The composition of any one of claims 2-8, wherein the targeting moiety comprises an aptamer.

[0175] 10. The composition of any one of claims 2 to 9, wherein the targeting moiety comprises a nucleic acid.

[0176] 11. The composition of any one of claims 2 to 10, wherein the targeting moiety comprises an antibody or a fragment thereof.

[0177] 12. The composition of any one of claims 2-11, wherein the targeting moiety comprises a nanobody, unibody, minibody, diabody, triabody, and / or tetrabody.

[0178] 13. The composition of any one of claims 2-12, wherein the targeting moiety comprises an organic molecule.

[0179] 14. The composition of any one of claims 2 to 13, wherein the targeting moiety comprises a ligand.

[0180] 15. The composition of any one of claims 2-14, wherein the targeting moiety comprises a cell-penetrating peptide.

[0181] 16. The composition of claim 15, wherein the cell-penetrating peptide is chemically conjugated to the polysialic acid.

[0182] 17. The composition of any one of claims 2-16, wherein the targeting moiety comprises a CendR peptide.

[0183] 18. The targeting moiety is the amino acid sequence Z 1 X 1 X 2 Z 2 Contains Z 1 is R or K, and Z 2 is R or K, and X 1 and X 2 and each represent an amino acid residue.

[0184] 19. The composition of any one of claims 2 to 18, wherein the targeting moiety comprises the amino acid sequence RGD.

[0185] 20. The composition of any one of claims 2 to 19, wherein the targeting moiety comprises the amino acid sequence NGR.

[0186] 21. The targeting moiety is the amino acid sequence CJ 1 Z 1 X 1 X 2 Z 2 Including J 1 21. The composition according to claim 2, wherein: is an amino acid sequence.

[0187] 22. The targeting moiety is the amino acid sequence J 1 Contains RGD, J 1 22. The composition according to claim 2, wherein: is an amino acid sequence.

[0188] 23. The targeting moiety is the amino acid sequence J 1 Z 1 X 1 X 2 Z 2 J 2 Including J 1 and J. 223. The composition according to claim 2, wherein each of the following is independently an amino acid sequence.

[0189] 24. The targeting moiety is the amino acid sequence J 1 RGDJ 2 Including J 1 and J. 2 24. The composition according to claim 2, wherein each of the following is independently an amino acid sequence.

[0190] 25. The targeting moiety is the amino acid sequence CJ 1 Z 1 X 1 X 2 Z 2 J 2 Including J 1 and J. 2 25. The composition according to claim 2, wherein each of the following is independently an amino acid sequence.

[0191] 26. The composition of any one of claims 2-25, wherein the targeting moiety comprises Lyp-1.

[0192] 27. The composition of any one of claims 2-26, wherein the targeting moiety comprises tLyp-1.

[0193] 28. The composition of any one of claims 2 to 27, wherein the targeting moiety comprises cLyp1.

[0194] 29. The composition of any one of claims 2-28, wherein the targeting moiety comprises iNGR.

[0195] 30. The composition of any one of claims 2-29, wherein the targeting moiety comprises iRGD.

[0196] 31. The composition of any one of claims 2-30, wherein the targeting moiety comprises an RPARPAR.

[0197] 32. The composition of any one of claims 2-31, wherein the targeting moiety comprises TT1.

[0198] 33. The composition of any one of claims 2-32, wherein the targeting moiety comprises linear TT1.

[0199] 34. The composition of any one of claims 2-33, wherein the targeting moiety comprises RGD-4C.

[0200] 35. The composition of any one of claims 2-34, wherein the targeting moiety comprises cRGD.

[0201] 36. The composition of any one of claims 2-35, wherein the targeting moiety comprises cilengitide.

[0202] 37. The composition of any one of claims 2-36, wherein the targeting moiety is selected from the group consisting of Lyp1, tLyp1, cLyp1, iNGR, iRGD, RPARPAR, TT1, linear TT1, RGD-4C, cRGD, cilengitide, F3, 9-RGD, RGD4C, delta24-RGD, delta24-RGD4C, RGD-K5, acyclic RGD4C, bicyclic RGD4C, c(RGDfK), c(RGDyK), E-[c(RGDfK)2], E[c(RGDyK)]2, KLWVLPKGGGC, CDCRGDCFC, LABL, angiopeptin-2, an antibody, a nanobody, transferrin, ankyrin repeat protein, an affibody, folate, triphenylphosphonium, ACUPA, PSMA, a carbohydrate moiety, and an aptamer.

[0203] 38. The composition of any one of claims 1-37, wherein the shell further comprises a penetration enhancer.

[0204] 39. The composition of any one of claims 1-38, wherein at least a portion of the polysialic acid is conjugated to a hydrophobic moiety.

[0205] 40. The composition of claim 39, wherein the hydrophobic moiety is selected from alkyl groups, cycloalkanes, bile salts and derivatives, terpenoids, terpenes, terpene-derived moieties, and fat-soluble vitamins.

[0206] 41. The composition of any one of claims 39 or 40, wherein the hydrophobic moiety comprises a straight-chain alkyl group.

[0207] 42. The composition of any one of claims 39-41, wherein the hydrophobic moiety contains at least two carbon atoms.

[0208] 43. The composition of any one of claims 39-42, wherein the hydrophobic moiety contains at least 3 carbon atoms.

[0209] 44. Hydrophobic part C2-C 24 44. The composition of any one of claims 39 to 43, comprising a straight chain alkyl group.

[0210] 45. Hydrophobic part is linear C 12 45. The composition of any one of claims 39 to 44, comprising an alkyl group.

[0211] 46. ​​The composition of any one of claims 1-45, further comprising an aliphatic carbon chain covalently bonded to the polysialic acid.

[0212] 47. Aliphatic carbon chain: C2-C 24 47. The composition of claim 46, comprising an aliphatic carbon chain.

[0213] 48. The composition of any one of claims 1-47, wherein at least about 90% by weight of the shell comprises polysialic acid.

[0214] 49. The composition of any one of claims 1-48, wherein at least a portion of the plurality of nano-entities is substantially solid.

[0215] 50. The composition of any one of claims 1-49, wherein at least some of the plurality of nano-entities are nanocapsules.

[0216] 51. The composition of any one of claims 1-50, wherein at least a portion of the polysialic acid comprises N-acetylneuraminic acid.

[0217] 52. The composition of any one of claims 1-51, wherein at least a portion of the polysialic acid comprises 2-keto-3-deoxynonic acid.

[0218] 53. The composition of any one of claims 1-52, wherein at least a portion of the polysialic acid comprises lactamic acid.

[0219] 54. The composition of any one of claims 1-53, wherein at least a portion of the polysialic acid comprises N-sialic acid.

[0220] 55. The composition of any one of claims 1-54, wherein at least a portion of the polysialic acid comprises O-sialic acid.

[0221] 56. The composition of any one of claims 1-55, wherein at least a portion of the polysialic acid comprises at least two sialic acid units.

[0222] 57. The composition of any one of claims 1-56, wherein at least a portion of the polysialic acid comprises at least four sialic acid units.

[0223] 58. The composition of any one of claims 1-57, wherein at least a portion of the polysialic acid comprises at least 8 sialic acid units.

[0224] 59. The composition of any one of claims 1-58, wherein at least a portion of the polysialic acid comprises sialic acid units linked via 2-->8 linkages.

[0225] 60. The composition of any one of claims 1-59, wherein at least a portion of the polysialic acid comprises sialic acid units linked via 2-->9 linkages.

[0226] 61. The composition of any one of claims 1-60, wherein the inner portion is non-aqueous.

[0227] 62. The composition of any one of claims 1-61, wherein the inner portion comprises a pharmaceutical agent.

[0228] 63. The composition of claim 62, wherein the pharmaceutical agent is lipid-soluble.

[0229] 64. The composition of claim 62, wherein the pharmaceutical agent is amphiphilic.

[0230] 65. The composition of claim 62, wherein the pharmaceutical agent is water-soluble.

[0231] 66. The composition of claim 62, wherein the pharmaceutical agent is a monoclonal antibody.

[0232] 67. The composition of claim 62, wherein the pharmaceutical agent is a polynucleotide.

[0233] 68. The composition of claim 62, wherein the pharmaceutical agent is docetaxel.

[0234] 69. The composition of claim 62, wherein the pharmaceutical agent is an anti-cancer agent.

[0235] 70. The drug is gemcitabine, paclitaxel, cabazitaxel, tomudex, daunomycin, aclarubicin, bleomycin, dactinomycin, daunorubicin, rapamycin, epirubicin, valrubicin, idarubicin, mitomycin C, mitoxantrone, elesclomol, ingenol mebutate, plicamycin, calicheamicin, esperamicin, degarelix, emtansine, maytansine, maytansinoid DM1, maytansinoid 2, maytansinoid DM4, mitomycin, auristatin, vinorelbine, vinblastine, vincristine, vindesine, estramustine, hydrophobic derivatives of cisplatin, chlorambucil, bendamustine, carmustine, amantadine, rimantadine, lomustine, semustine, amsacrine, ladribine, cytarabine, (C 12 ~C 18)-Gemcitabine, tegafur, trimetrexate, sagopilone, ixapebilone, patupilone, eribulin, camptothecin, aminoglutethimide, diaziquone, levamisole, methyl-GAG, mitotane, mitoxantrone, testolactone, michelanin B, bryostatin-1, halomon, didemnin, plitidepsin, trabectedin, lurbinectedin, vorinostat, romidepsin, ibuprofen Rinotecan, bortezomib, erlotinib, getifinib, imatinib, vemurafenib, crizotinib, vismodegib, tretinoin, alitretinoin, bexarotene, tacrolimus, everolimus, topotecan, teniposide, etoposide, pralatrexate, omacetaxine, doxorubicin, dacarbazine, procarbazine, hydroxydaunorubicin, hydroxyurea, 6-mercaptopurine, 6-thioguanine, floxuridine or 5-fluorodeoxyuridine, fludarabine, 5-fluorouracil, methotrexate, thiotepa, pentostatin, mechlorethamine, pibobroman, cyclophosphamide, ifosfamide, busulfan, carboplatin, picoplatin, tetraplatin, satrapalin, platinum-DACH, ormaplatin, oxaplatin, metoprolol, methylparaben ... 70. The composition of claim 69, wherein the compound is selected from the group consisting of ruffalin, aminoglutethimide, trastuzumab, bevazizumab, durvalumab, nivolumab, inotuzumab, avelumab, pembrolizumab, olaratumumab, atezolizumab, daratumumab, elotuzumab, necitumumab, dinutuximab, blinatumomab, ramucirumab, obinutuzumab, denosumab, ipilimumab, brentuximab, ofatumumab, and combinations thereof.

[0236] 71. The composition of any one of claims 62-70, wherein the inner portion comprises at least two pharmaceutical agents.

[0237] 72. The composition of any one of claims 1-71, wherein the shell contains a pharmaceutical agent.

[0238] 73. The composition of claim 72, wherein the drug substance in the shell is fat-soluble.

[0239] 74. The composition of claim 72, wherein the drug substance in the shell is amphiphilic.

[0240] 75. The composition of claim 72, wherein the pharmaceutical agent in the shell is water-soluble.

[0241] 76. The composition of claim 72, wherein the outer shell pharmaceutical agent is a polynucleotide.

[0242] 77. The composition of claims 1-76, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0243] 78. The composition of any one of claims 1-77, wherein the plurality of nano-entities have an average diameter of less than 250 nm.

[0244] 79. The composition of any one of claims 1-78, wherein the plurality of nano-entities have an average diameter of less than 150 nm.

[0245] 80. The composition of any one of claims 1-79, wherein the plurality of nano-entities comprises micelles.

[0246] 81. The composition of any one of claims 1-80, wherein the plurality of nano-entities are not liposomes.

[0247] 82. The composition of any one of claims 1-81, wherein the plurality of nano-entities does not comprise protamine.

[0248] 83. The composition of any one of claims 1-82, wherein the plurality of nano-entities does not comprise polyarginine.

[0249] 84. The composition of any one of claims 1-83, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0250] 85. (Aspect 2): A composition according to any one of claims 1 to 84, for use as a medicament.

[0251] 86. A method comprising administering to an organism the composition of any one of claims 1-85.

[0252] 87. The method of claim 86, wherein the organism is a human.

[0253] 88. (Embodiment 3): A method comprising reacting carboxylate moieties on a polysialic acid with an aminoalkyl(C1-C4)maleimide and / or aminoalkyl(C1-C4)methacrylamide; and reacting the resulting aminoalkyl(C1-C4)maleimide and / or aminoalkyl(C1-C4)methacrylamide with thiol groups on a targeting moiety to obtain a polysialic acid-aminoalkyl(C1-C4)succinimide-peptide and / or polysialic acid-aminoalkyl(C1-C4)amide isopropyl-peptide composition.

[0254] 89. The method of claim 88, further comprising: forming an emulsion comprising the polysialic acid-aminoalkyl(C1-C4) succinimide-peptide composition and / or the polysialic acid-aminoalkyl(C1-C4) amidoisopropyl-peptide composition; and forming a plurality of nanoparticles from the emulsion.

[0255] 90. The method of claim 89, wherein at least a portion of the plurality of nanoparticles comprises an inner portion surrounded by an exposed outer shell.

[0256] 91. (Embodiment 4): A method comprising: reacting a carboxylate moiety on a polysialic acid with N-hydroxysuccinimide and / or a carbodiimide to form an intermediate; and reacting the intermediate with a lysine or arginine group on a targeting moiety to obtain a polysialic acid-amide-peptide.

[0257] 92. The method of claim 91, further comprising: forming an emulsion comprising the polysialic acid-amide-peptide; and forming a plurality of nanoparticles from the emulsion.

[0258] 93. The method of claim 92, wherein at least a portion of the plurality of nanocapsules comprises an interior portion surrounded by an exposed outer shell.

[0259] 94. (Embodiment 5): A composition comprising a plurality of nanoentities comprising an inner portion surrounded by an outer shell, the outer shell comprising polysialic acid, and at least some of the nanoentities further comprising a monoclonal antibody contained in the inner portion.

[0260] 95. The composition of claim 94, wherein the monoclonal antibody is not exposed on the exterior of the nano-entity.

[0261] 96. The composition of any one of claims 94 or 95, wherein at least a portion of the plurality of nano-entities further comprise one or more surfactants.

[0262] 97. The composition of any one of claims 94-96, wherein at least a portion of the plurality of nano-entities further comprise a targeting moiety and / or a cell-penetrating peptide and / or a tumor / tissue-penetrating peptide.

[0263] 98. The composition of claim 97, wherein the targeting moiety is electrostatically bound to the polysialic acid.

[0264] 99. The composition of claim 97, wherein the targeting moiety is attached to the polysialic acid via a linker.

[0265] 100. The composition of claim 97, wherein the targeting moiety is attached to the polysialic acid via an aminoalkyl(C1-C4) succinimide linker, an aminoalkyl(C1-C4) amido-iso-propyl linker, or directly through the amide group.

[0266] 101. The composition of claim 100, wherein the aminoalkyl (C1-C4) succinimide linker is generated by an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) coupling reaction.

[0267] 102. The composition of claim 99, wherein the targeting moiety is attached to the polysialic acid via an aminoethylsuccinimide linker.

[0268] 103. The composition of any one of claims 97-102, wherein the targeting moiety comprises a cell-penetrating peptide.

[0269] 104. The composition of claim 103, wherein the cell-penetrating peptide is chemically conjugated to the polysialic acid.

[0270] 105. The composition of any one of claims 97-104, wherein the targeting moiety comprises the amino acid sequence RGD.

[0271] 106. The composition of any one of claims 97-105, wherein the targeting moiety comprises the amino acid sequence NGR.

[0272] 107. The composition of any one of claims 97-106, wherein the targeting moiety comprises Lyp-1.

[0273] 108. The composition of any one of claims 97-107, wherein the targeting moiety comprises tLyp-1.

[0274] 109. The composition of any one of claims 97-108, wherein the targeting moiety comprises cLyp1.

[0275] 110. The composition of any one of claims 94-109, wherein the shell further comprises a penetration enhancer.

[0276] 111. The composition of any one of claims 94-109, wherein at least a portion of the polysialic acid is conjugated to a hydrophobic moiety.

[0277] 112. The composition of any one of claims 94-111, wherein at least about 93% by weight of the shell comprises polysialic acid.

[0278] 113. The composition of any one of claims 94-112, wherein at least some of the plurality of nano-entities are nanocapsules.

[0279] 114. The composition of any one of claims 94-113, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0280] 115. The composition of any one of claims 94-114, wherein the plurality of nano-entities have an average diameter of less than 250 nm.

[0281] 116. The composition of any one of claims 94-115, wherein the plurality of nano-entities have an average diameter of less than 150 nm.

[0282] 117. The composition of any one of claims 94-116, wherein the plurality of nano-entities are not liposomes.

[0283] 118. The composition of any one of claims 94-117, wherein the plurality of nano-entities does not comprise protamine.

[0284] 119. The composition of any one of claims 94-118, wherein the plurality of nano-entities does not comprise polyarginine.

[0285] 120. The composition of any one of claims 94-119, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0286] 121. (Embodiment 6): A composition according to any one of claims 94 to 120, for use as a medicament.

[0287] 122. A method comprising administering to an organism the composition of any one of claims 94-120.

[0288] 123. (Embodiment 7): A composition comprising a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell consisting essentially of polysialic acid, and the inner portion comprising at least one hydrophobic compound.

[0289] 124. The composition of claim 123, wherein the shell is at least 90% by weight polysialic acid.

[0290] 125. The composition of any one of claims 123 or 124, wherein at least a portion of the plurality of nano-entities further comprise a surfactant located between the interior portion and the outer shell.

[0291] 126. The composition of any one of claims 123-125, wherein at least a portion of the plurality of nano-entities further comprise a targeting moiety comprising a cell-penetrating peptide chemically conjugated to polysialic acid.

[0292] 127. The composition of claim 126, wherein the targeting moiety comprises a CendR peptide.

[0293] 128. The composition of any one of claims 126 or 127, wherein the targeting moiety comprises tLyp-1.

[0294] 129. The composition of any one of claims 126-128, wherein the targeting moiety is attached to the polysialic acid via a linker.

[0295] 130. The composition of any one of claims 126-129, wherein the targeting moiety is attached to the polysialic acid via an aminoalkyl(C1-C4) succinimide linker.

[0296] 131. The composition of any one of claims 123-130, wherein the plurality of nano-entities are not liposomes.

[0297] 132. The composition of any one of claims 123-131, wherein the plurality of nano-entities does not comprise protamine.

[0298] 133. The composition of any one of claims 123-132, wherein the plurality of nano-entities does not comprise polyarginine.

[0299] 134. The composition of any one of claims 123-133, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0300] 135. (Embodiment 8): A composition according to any one of claims 123 to 134, for use as a medicament.

[0301] 136. A method comprising administering to an organism the composition of any one of claims 123-134.

[0302] 137. (Embodiment 9): A composition comprising a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell comprising polysialic acid and a targeting moiety comprising a cell-penetrating peptide chemically bound to the polysialic acid.

[0303] 138. The composition of claim 137, wherein the plurality of nano-entities are not liposomes.

[0304] 139. (Aspect 10): A composition according to any one of claims 137 or 138 for use as a medicament.

[0305] 140. A method comprising administering the composition of any one of claims 137 or 138 to an organism.

[0306] 141. (Embodiment 11): An antibody comprising an inner portion surrounded by an outer shell, the outer shell comprising polysialic acid and a targeting moiety chemically bound to the polysialic acid, the targeting moiety having the sequence Z 1 X 1 X 2 Z 2 and / or a peptide having the sequence RGD and / or the sequence NGR, Z 1 is R or K, and Z 2 is R or K, and X 1 and X 2 is an amino acid residue.

[0307] 142. The composition of claim 141, wherein the plurality of nano-entities are not liposomes.

[0308] 143. (Aspect 12): A composition according to any one of claims 141 or 142 for use as a medicament.

[0309] 144. A method comprising administering the composition of any one of claims 141 or 142 to an organism.

[0310] 145. (Aspect 13): A composition comprising a plurality of entities having a maximum average diameter of less than about 1 micrometer, the entities having a surface comprising polysialic acid and a targeting moiety, with the proviso that the entities are not liposomes.

[0311] 146. The composition of claim 145, wherein at least some of the plurality of nano-entities are nanocapsules.

[0312] 147. The composition of any one of claims 145 or 146, wherein at least a portion of the plurality of nano-entities are micelles.

[0313] 148. The composition of any one of claims 145-147, wherein the targeting moiety comprises a cell-penetrating peptide.

[0314] 149. The composition of any one of claims 145-148, wherein the plurality of entities is not a liposome.

[0315] 150. (Embodiment 14): A composition according to any one of claims 145 to 149 for use as a medicament.

[0316] 151. A method comprising administering to an organism the composition of any one of claims 145-149.

[0317] 152. (Embodiment 15): A kit comprising a composition according to any one of claims 1 to 84, 94 to 120, 123 to 134, 137, 138, 141, 142, or 145 to 149.

[0318] 153. (Aspect 16): A composition comprising a plurality of nanoentities, each nanoentity comprising an inner portion surrounded by an outer shell, the outer shell comprising hyaluronic acid, and at least some of the nanoentities further comprising a monoclonal antibody.

[0319] 154. The composition of claim 153, wherein at least about 90% by weight of the shell comprises hyaluronic acid.

[0320] 155. The composition of any one of claims 153 or 154, wherein at least some of the plurality of nano-entities are nanocapsules.

[0321] 156. The composition of any one of claims 153-155, wherein the inner portion is non-aqueous.

[0322] 157. The composition of any one of claims 153-156, wherein the monoclonal antibody is contained within the inner portion.

[0323] 158. The composition of any one of claims 153-157, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0324] 159. The composition of any one of claims 153-158, wherein the nanoentity is a micelle.

[0325] 160. The composition of any one of claims 153-159, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0326] 161. (Embodiment 17): A composition according to any one of claims 153 to 160 for use as a medicament.

[0327] 162. (Embodiment 18): A composition comprising a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell comprising PGA and / or PASP and a targeting moiety.

[0328] 163. The composition of claim 162, wherein the targeting moiety is electrostatically bound to PGA and / or PASP.

[0329] 164. The composition of any one of claims 162 or 163, wherein the targeting moiety is attached to PGA and / or PASP via a linker.

[0330] 165. The composition of any one of claims 162 or 163, wherein the targeting moiety is attached to the PGA and / or PASP via an aminoalkyl(C1-C4)maleimide linker, an aminoalkyl(C1-C4)methacrylamide linker, or directly through an amide group.

[0331] 166. The composition of claim 165, wherein the targeting moiety is attached to PGA and / or PASP via an aminoethylmaleimide linker.

[0332] 167. The composition of any one of claims 162-166, wherein the targeting moiety comprises a cell-penetrating peptide.

[0333] 168. The composition of any one of claims 162-167, wherein the cell-penetrating peptide is chemically conjugated to PGA and / or PASP.

[0334] 169. The composition of any one of claims 162-168, wherein the targeting moiety comprises a CendR peptide.

[0335] 170. The composition of any one of claims 162-169, wherein the targeting moiety comprises Lyp-1.

[0336] 171. The composition of any one of claims 162-170, wherein the targeting moiety comprises tLyp-1.

[0337] 172. The composition of any one of claims 162-171, wherein the targeting moiety comprises cLyp1.

[0338] 173. The composition of any one of claims 162-172, wherein at least about 90% by weight of the shell comprises PGA and / or PASP.

[0339] 174. The composition of any one of claims 162-173, wherein at least some of the plurality of nano-entities are nanocapsules.

[0340] 175. The composition of any one of claims 162-174, wherein the inner portion is non-aqueous.

[0341] 176. The composition of any one of claims 162-175, wherein the inner portion comprises a pharmaceutical agent.

[0342] 177. The composition of claim 176, wherein the pharmaceutical agent is a monoclonal antibody.

[0343] 178. The composition of any one of claims 162-177, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0344] 179. The composition of any one of claims 162-178, wherein the nanoentity is a micelle.

[0345] 180. The composition of any one of claims 162-179, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0346] 181. The composition of any one of claims 162-180, wherein at least a portion of the PGA and / or PASP is associated with a hydrophobic moiety.

[0347] 182. (Embodiment 19): A composition according to any one of claims 162 to 181, for use as a medicament.

[0348] 183. (Embodiment 20): An antibody comprising an inner portion surrounded by an outer shell, the outer shell comprising PGA and / or PASP and a targeting moiety, the targeting moiety comprising sequence Z 1 X 1 X 2 Z 2 and / or a peptide having the sequence RGD and / or the sequence NGR, Z 1 is R or K, and Z 2 is R or K, and X 1 and X 2 is an amino acid residue.

[0349] 184. The composition of claim 183, wherein the targeting moiety is electrostatically bound to PGA and / or PASP.

[0350] 185. The composition of any one of claims 183 or 184, wherein the targeting moiety is attached to PGA and / or PASP via a linker.

[0351] 186. The composition of any one of claims 183 or 185, wherein the targeting moiety is attached to the PGA and / or PASP via an aminoalkyl(C1-C4)maleimide linker, an aminoalkyl(C1-C4)methacrylamide linker, or directly through an amide group.

[0352] 187. The composition of claim 186, wherein the targeting moiety is attached to PGA and / or PASP via an aminoethylmaleimide linker.

[0353] 188. The composition of any one of claims 183-187, wherein at least about 90% by weight of the shell comprises PGA and / or PASP.

[0354] 189. The composition of any one of claims 183-188, wherein at least some of the plurality of nano-entities are nanocapsules.

[0355] 190. The composition of any one of claims 183-189, wherein the inner portion is non-aqueous.

[0356] 191. The composition of any one of claims 183-190, wherein the inner portion comprises a pharmaceutical agent.

[0357] 192. The composition of claim 191, wherein the pharmaceutical agent is a monoclonal antibody.

[0358] 193. The composition of any one of claims 183-192, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0359] 194. The composition of any one of claims 183-193, wherein the nanoentity is a micelle.

[0360] 195. The composition of any one of claims 183-194, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0361] 196. The targeting moiety is the amino acid sequence CJ 1 Z 1 X 1 X 2 Z 2 Including J 1 The composition of any one of claims 183 to 195, wherein is an amino acid sequence.

[0362] 197. The targeting moiety is the amino acid sequence J 1 Contains RGD, J 1 The composition of any one of claims 183 to 196, wherein is an amino acid sequence.

[0363] 198. The targeting moiety is the amino acid sequence J 1 Z 1 X 1 X 2 Z 2 J 2 Including J 1 and J. 2 The composition of any one of claims 183 to 197, wherein each of the is independently an amino acid sequence.

[0364] 199. The targeting moiety is the amino acid sequence J 1 RGDJ 2 Including J 1 and J. 2 The composition of any one of claims 183 to 198, wherein each of the is independently an amino acid sequence.

[0365] 200. The targeting moiety is the amino acid sequence CJ 1 Z 1 X 1 X 2 Z 2 J 2 Including J 1 and J. 2200. The composition of any one of claims 183 to 199, wherein each of the is independently an amino acid sequence.

[0366] 201. (Embodiment 21): A composition according to any one of claims 183 to 200, for use as a medicament.

[0367] 202. (Embodiment 22): A composition comprising a plurality of nanoentities comprising an inner portion surrounded by an outer shell, the outer shell comprising PGA and / or PASP, and at least some of the nanoentities further comprising a monoclonal antibody contained within the inner portion.

[0368] 203. The composition of claim 202, wherein at least about 90% by weight of the shell comprises PGA and / or PASP.

[0369] 204. The composition of any one of claims 202 or 203, wherein at least some of the plurality of nano-entities are nanocapsules.

[0370] 205. The composition of any one of claims 202-204, wherein the inner portion is non-aqueous.

[0371] 206. The composition of any one of claims 202-205, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0372] 207. The composition of any one of claims 202-206, wherein the nanoentity is a micelle.

[0373] 208. The composition of any one of claims 202-207, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0374] 209. (Embodiment 23): A composition according to any one of claims 202 to 208 for use as a medicament.

[0375] 210. (Embodiment 24): A composition comprising a plurality of nano-entities comprising an inner portion surrounded by an outer shell, the outer shell comprising hyaluronic acid bound to a hydrophobic portion.

[0376] 211. The composition of claim 210, wherein at least some of the nano-entities further comprise a monoclonal antibody contained within the interior portion.

[0377] 212. The composition of any one of claims 210 or 211, wherein at least some of the nano-entities further comprise a pharmaceutical agent contained within the interior portion.

[0378] 213. The composition of any one of claims 210-212, wherein at least some of the nano-entities further comprise a small molecule contained within the interior portion.

[0379] 214. The composition of any one of claims 210-213, wherein the hydrophobic moiety is selected from alkyl groups, cycloalkanes, bile salts and derivatives, terpenoids, terpenes, terpene-derived moieties, and fat-soluble vitamins.

[0380] 215. The composition of any one of claims 210-214, wherein the hydrophobic moiety comprises a straight-chain alkyl group.

[0381] 216. Hydrophobic part: C2-C 24 216. The composition of any one of claims 210-215, comprising a straight chain alkyl group.

[0382] 217. Hydrophobic part is linear C 16 217. The composition of any one of claims 210-216, comprising an alkyl group.

[0383] 218. The composition of any one of claims 210-217, wherein at least a portion of the plurality of nano-entities further comprise a targeting moiety.

[0384] 219. The composition of claim 218, wherein the targeting moiety comprises Lyp-1.

[0385] 220. The composition of any one of claims 218 or 219, wherein the targeting moiety comprises tLyp-1.

[0386] 221. The composition of any one of claims 218-220, wherein the targeting moiety comprises cLyp1.

[0387] 222. The composition of any one of claims 218-221, wherein the targeting moiety comprises a cell-penetrating peptide.

[0388] 223. The composition of any one of claims 210-222, wherein at least about 90% by weight of the shell comprises hyaluronic acid.

[0389] 224. The composition of any one of claims 210-223, wherein at least some of the plurality of nano-entities are nanocapsules.

[0390] 225. The composition of any one of claims 210-224, wherein the inner portion is non-aqueous.

[0391] 226. The composition of any one of claims 210-225, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0392] 227. The composition of any one of claims 210-226, wherein the nanoentity is a micelle.

[0393] 228. The composition of any one of claims 210-227, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0394] 229. (Embodiment 25): A composition according to any one of claims 210 to 228, for use as a medicament.

[0395] 230. (Embodiment 26): A composition comprising a plurality of nanoentities, each nanoentity comprising an inner portion surrounded by an outer shell, the outer shell comprising a polymer selected from the group consisting of a polyacid, a polyester, a polyamide, or a mixture thereof, and at least some of the nanoentities further comprising a monoclonal antibody.

[0396] 231. The composition of claim 230, wherein the monoclonal antibody is contained within the interior portion.

[0397] 232. The composition of any one of claims 230 or 231, wherein at least about 90% by weight of the shell comprises a polymer.

[0398] 233. The composition of any one of claims 230-232, wherein the polymer comprises polysialic acid.

[0399] 234. The composition of any one of claims 230-233, wherein the polymer comprises hyaluronic acid.

[0400] 235. The composition of any one of claims 230-234, wherein the polymer comprises polyglutamic acid and / or PGA-PEG.

[0401] 236. The composition of any one of claims 230-235, wherein the polymer comprises PASP and / or PASP-PEG.

[0402] 237. The composition of any one of claims 230-236, wherein the polymer comprises polylactic acid-polyethylene glycol (PLA-PEG).

[0403] 238. The composition of any one of claims 230-237, wherein the polymer comprises poly(lactic-co-glycolic acid) and / or pegylated poly(lactic-co-glycolic acid).

[0404] 239. The composition of any one of claims 230-238, wherein the polymer comprises polylactic acid and / or pegylated polylactic acid.

[0405] 240. The composition of any one of claims 230-239, wherein the polymer comprises polyaspartic acid and / or pegylated polyaspartic acid.

[0406] 241. The composition of any one of claims 230-240, wherein the polymer comprises alginic acid and / or pegylated alginic acid.

[0407] 242. The composition of any one of claims 230-241, wherein the polymer comprises polymalic acid and / or PEGylated polymalic acid.

[0408] 243. The composition of any one of claims 230-242, wherein the polymer is conjugated to a hydrophobic moiety.

[0409] 244. The composition of any one of claims 230-243, wherein at least some of the nano-entities further comprise a targeting moiety.

[0410] 245. The composition of any one of claims 230-244, wherein at least some of the plurality of nano-entities are nanocapsules.

[0411] 246. The composition of any one of claims 230-245, wherein the inner portion is non-aqueous.

[0412] 247. The composition of any one of claims 230-246, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0413] 248. The composition of any one of claims 230-247, wherein the nanoentity is a micelle.

[0414] 249. The composition of any one of claims 230-248, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0415] 250. (Embodiment 27): A composition according to any one of claims 230 to 249 for use as a medicament.

[0416] 251. (Aspect 28): A composition comprising a plurality of nanoentities, each nanoentity comprising an inner portion surrounded by an outer shell, the outer shell comprising hyaluronic acid bound to a hydrophobic moiety, and at least a portion of the nanoentities further comprising a small molecule having a molecular weight of less than 1000 Da.

[0417] 252. The composition of claim 251, wherein the small molecule is contained within the inner portion.

[0418] 253. The composition of any one of claims 251 or 252, wherein the small molecule is a pharmaceutical agent.

[0419] 254. The composition of any one of claims 251-153, wherein the small molecule is docetaxel.

[0420] 255. The composition of any one of claims 251-254, wherein the hydrophobic moiety is selected from alkyl groups, cycloalkanes, bile salts and derivatives, terpenoids, terpenes, terpene-derived moieties, and fat-soluble vitamins.

[0421] 256. The composition of any one of claims 251-255, wherein the hydrophobic moiety comprises a straight-chain alkyl group.

[0422] 257. Hydrophobic part C2-C 24 257. The composition of any one of claims 251-256, comprising a straight chain alkyl group.

[0423] 258. Hydrophobic part is linear C 16 258. The composition of any one of claims 251-257, comprising an alkyl group.

[0424] 259. The composition of any one of claims 251-258, wherein at least about 90% by weight of the shell comprises hyaluronic acid.

[0425] 260. The composition of any one of claims 251-259, wherein at least some of the plurality of nano-entities are nanocapsules.

[0426] 261. The composition of any one of claims 251-260, wherein at least some of the nano-entities further comprise a targeting moiety.

[0427] 262. The composition of any one of claims 251-261, wherein the targeting moiety comprises Lyp-1.

[0428] 263. The composition of any one of claims 251-262, wherein the targeting moiety comprises tLyp-1.

[0429] 264. The composition of any one of claims 251-263, wherein the targeting moiety comprises cLyp1.

[0430] 265. The composition of any one of claims 251-264, wherein the targeting moiety comprises a cell-penetrating peptide.

[0431] 266. The composition of any one of claims 251-265, wherein the targeting moiety is conjugated to hyaluronic acid.

[0432] 267. The composition of any one of claims 251-266, wherein the inner portion is non-aqueous.

[0433] 268. The composition of any one of claims 251-267, wherein the plurality of nano-entities have an average diameter of less than 1 micrometer.

[0434] 269. The composition of any one of claims 251-268, wherein the nanoentity is a micelle.

[0435] 270. The composition of any one of claims 251-269, wherein the plurality of nano-entities does not comprise more than one outer shell.

[0436] 271. (Embodiment 29): A composition according to any one of claims 251 to 270 for use as a medicament.

[0437] 272. (Embodiment 30): A kit comprising a composition described in any one of claims 153-160, 162-181, 183-200, 202-208, 210-2298, 230-249, or 251-270.

[0438] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0439] Example 1 This example describes polysialic acid (PSA) nanocapsules that are functionalized or not with the tumor-penetrating peptide tLyp1.

[0440] The nanocapsules had the following composition: They were formed from an oily core surrounded by a polymeric shell of PSA or PSA functionalized with tLyp1 peptide and stabilized by a surfactant. The nanocapsules were formed by the interaction of PSA with a positively charged surfactant at the interphase of an oil-in-water emulsion. Unless otherwise specified, the PSA used had a molecular weight of approximately 30 kDa (26–30 kDa, Serum Institute of India).

[0441] The covalent bond of PSA used allows selective covalent conjugation between the thiol group of the peptide tLyp1 and the carboxylate group of PSA. This synthetic method uses a heterobifunctional linker, aminoethylmaleimide, which allows peptide conjugation in a two-step process: first, incorporation of the amine group of the linker into the carboxylate group of PSA (using carbodiimide chemistry), and then, via Michael-type addition of the thiol group (cysteine ​​residue) of the peptide to the maleimide group of the linker (Figure 1). This strategy allows for protection of the biologically active groups of the tLyp1 peptide. Furthermore, the degree of substitution can be easily controlled.

[0442] Polymer nanocapsules, such as PSA nanocapsules, can be fabricated by various techniques. The number of tLyp1 molecules on the nanocapsule surface could be modified by varying the molar ratio used in the chemical reaction (see Table 1, which shows the molar feed ratios of PSA, EDC, NHS, and carboxylic acid (COOH)). One such technique is the solvent displacement technique, in which a polar solvent is mixed in the aqueous phase. Another technique is the self-emulsification technique, which does not require the use of organic solvents.

[0443] Polymer nanocapsules, such as PSA nanocapsules, were successfully functionalized with tLyp1. The number of tLyp1 molecules on the nanocapsule surface could be controlled. The formed tLyp1-functionalized nanocapsules were approximately 130 nm in size and had a negative zeta potential (-44 mV). The tLyp1-functionalized nanocapsules were found to be stable even when incubated in plasma at 37°C. Furthermore, tLyp1-functionalized nanocapsules could be loaded with any suitable hydrophobic drug, as well as water-soluble molecules. In one experiment, tLyp1-functionalized nanocapsules were loaded with docetaxel, e.g., anhydrous docetaxel (MW 807.289 g / mol; LogP 2.6). In addition to tLyp1, other tissue-penetrating peptides, such as CendR peptides (e.g., Lyp1 and iRGD), could be attached to PSA chains.

[0444] PSA was modified with N-(2-aminoethyl)maleimide trifluoroacetate. Various molar ratios between the carboxylic acid groups of PSA and EDC, NHS, AEM, and tLyp1 were tested (Table 1). For this purpose, PSA was dissolved in 0.1 M MES buffer at pH 6 to a final concentration of 2 mg / mL. Corresponding amounts of EDC, NHS, and AEM were also dissolved in 0.1 M MES buffer, added to the PSA solution, and maintained at room temperature for 4 h under magnetic stirring. Maleimide-functionalized PSA (PSA-Mal) was purified by dialysis (regenerated cellulose, SnakeSkin 7 KDa MWCO, Thermo Scientific) first against 50 mM NaCl and then against MilliQ water. For the second reaction, PSA-Mal was dissolved in 0.1 M MES buffer and 50 mM NaCl to a final PSA concentration of 1 mg / mL. The peptide was added to this solution, the reaction mixture was kept under magnetic stirring at room temperature for 4 h, and the final PSA-tLyp1 product was purified by dialysis as previously described, lyophilized (Pilot Lyophilizer VirTis Genesis 25 ES), and stored at 4°C.

[0445] [Table 1]

[0446] PSA nanocapsules were prepared as follows. Nanocapsules with various ratios of PSA or PSA-tLyp1 polymer coating (e.g., with different Mw values ​​of 8 kDa, 26-30 kDa, and 94 kDa) were prepared by solvent displacement. The organic phase for docetaxel-loaded nanocapsules consisted of 4.75 mL of acetone and 0.25 mL of ethanol containing 0.75 mg / mL lecithin (Epikuron 145V, Cargill), 0.15 mg / mL cetyltrimethylammonium bromide (CTAB, Sigma-Aldrich), 2.96 mg / mL caprylic / capric triglyceride (Miglyol® 812, IOI Eleo), and 150 micrograms / mL docetaxel (Hao Rui Enterprises). The aqueous phase consisted of 10 mL of 0.25 mg / mL PSA or PSA-tLyp1 solution. The organic phase was added dropwise to the aqueous phase under magnetic stirring, resulting in the immediate formation of nanodroplets around which the polymer was deposited. After nanocapsule formation, the organic solvent was removed by rotary evaporation. Results are expressed as the mean + / - SD of three replicates (Table 2).

[0447] Nanocapsules with varying ratios of PSA or PSA-tLyp1 polymer coating were prepared using a Nanoassemblr® Benchtop microfluidics device (Precision Nanosystems) as follows: The aqueous phase consisted of 10 mL of a 0.25 mg / mL PSA or PSA-tLyp1 solution. The organic phase, for docetaxel-loaded nanocapsules, consisted of 1 mL of ethanol containing 3.75 mg Lipoid S100 (Lipoid), 0.75 mg benzethonium chloride (Spectrum Chemical), 15.3 mg Labrafac lipophile WL 1349 (Gattefosse), and 0.75 mg anhydrous docetaxel (Hao Rui Enterprises). Briefly, nanocapsules were fabricated by injecting both the aqueous and organic phases at adjustable flow rates into each inlet of the NanoAssemblr cartridge, where engineered microscopic features within the channel control rapid and uniform mixing of the two streams. After nanocapsule formation, ethanol was removed by rotary evaporation. Increasing the operating flow rate directly correlated with decreasing nanocapsule size (Table 3, PSA NC-A to C). Results are expressed as the mean + / - SD of three replicates (Table 3).

[0448] Nanocapsule isolation / concentration. Nanocapsules were isolated by ultracentrifugation (Optima™ L-90K Ultracentrifuge, Beckman Coulter; Fullerton, CA) at 84035 g and 15°C for 0.5 hours. The supernatant was then removed from the medium. The nanocapsules (supernatant) were collected and diluted to a known concentration.

[0449] Physicochemical characterization of nanocapsules. Nanocapsules were characterized in terms of mean particle size and polydispersity index (PI) by photon correlation spectroscopy (PCS). Samples were diluted with MilliQ water and analysis was performed at 25°C with a detection angle of 173°. Zeta potential measurements were performed by laser Doppler anemometry (LDA), with samples diluted with MilliQ ultrapure water. PCS and LDA analyses were performed in triplicate using a NanoZS® (Malvern Instruments, Malvern, UK).

[0450] Docetaxel conjugation efficiency (AE%). Docetaxel conjugation efficiency was expressed as the percentage of encapsulated drug in terms of the total docetaxel amount. Therefore, the drug encapsulated in an aliquot of isolated nanocapsules was determined, and the total drug amount in an aliquot of non-isolated nanocapsules was estimated. Drug quantification was performed by UPLC or liquid chromatography / tandem mass spectrometry (LC-MS) using paclitaxel as an internal standard. The UPLC system included an Acquity UPLC® H-class system (Waters) and a column compartment (BEH C18 column 2.1 × 100 mm, 1.7 micrometers, Waters). The experimental analysis conditions were as follows: the mobile phase included MilliQ water (A) and acetonitrile (B). An isocratic program of 55% A and 45% B was used. The flow rate was 0.4 mL / min, and the run time was 3.5 min. The column temperature was maintained at 40°C, and the autosampler was at a constant temperature of 4°C. The injection volume was 10 microliters. Under these conditions, DCX eluted at 1.8 + / - 0.02 minutes. The LC-MS system included a column compartment (BEH) coupled to a UPLC system (Acquity UPLC® H-Class System (Waters); Xevo® Triple Quadrupole Detector (TQD) (Waters, Milford, USA) equipped with an electrospray ionization (ESI) interface). A C18 column (2.1 x 100 mm, 1.7 micrometers, Waters) was included. Mass spectrometric detection was operated in positive mode and set to multiple reaction monitoring (MRM) to monitor the progression from m / z 830.4 to m / z 304.1 and from m / z 830.4 to m / z 549.2. The source temperature was selected to be 525 °C, the desolvation temperature to be 150 °C, the capillary voltage to be 3.1 kV, and the cone voltage to be 40 V. Nitrogen was used for the desolvation and cone gas at flow rates of 600 L / h and 80 L / h, respectively. Argon was used as the collision gas. The optimized collision energy was 30 eV. The experimental analytical conditions were as follows: the mobile phase included 0.1% formic acid in water (A) and acetonitrile (B).A linear gradient program was used, starting with 80% to 20% mobile phase A over 0-5 min, then returning to 80% A over 5-5.5 min and holding it constant until 6 min to reach initial conditions. The flow rate was 0.6 mL / min, with a total run time of 6 min. The column temperature was maintained at 40°C, and the autosampler was at a constant temperature of 4°C. The injection volume was 10 microliters. Under these conditions, DCX eluted at 4.11 + / - 0.02 min. Data collection and analysis were performed using TargetLynx v4.1 software (Waters).

[0451] [Table 2]

[0452] [Table 3]

[0453] Characterization of PSA-tlyp1 conjugates. Some NMR experiments were performed on a Varian Inova 750 spectrometer. Chemical shifts are reported in ppm. Spectra were recorded in a 10:90 mixture of deuterium oxide:MilliQ water at polymer concentrations of 0.4–0.8 mg / mL. 1 H-NMR analysis was performed at 750 MHz with 256 scans and a 10-second delay between scans. MestreNova Software (Mestrelab Research) was used to process the spectra. The peptide-derived characteristic features in the PSA-tLyp1 spectrum were identified. 1 The formation of the PSA-tLyp1 conjugate was confirmed by confirming the presence of a H-NMR signal. 1 The presence of signals characteristic of amine protons derived from amino acids of the tLyp1 peptide was observed at 6.5 to 8.5 ppm in the 1 H-NMR spectrum (Figure 13), thus confirming the covalent bond between PSA and tLyp1.

[0454] Example 2 This example presents in vivo data using the particles described in Example 1. Functionalization of PSA with tLyp1 resulted in a positive targeting effect in an orthotopic lung tumor model (high accumulation of the antitumor drug docetaxel in the lung). Biodistribution data shown in Figure 2A demonstrate that conjugation (e.g., covalent attachment) of the peptide to PSA significantly enhanced this targeting effect compared to administration of unconjugated tLyp1 (e.g., PSA nanocapsules and tLyp1 separately) and unmodified PSA nanocapsules (without tLyp1). Biodistribution data shown in Figure 2B demonstrate that the functionalized nanocapsules (PSA-tLyp1 NCs) accumulated approximately 26-fold more docetaxel in the tumor (lung) after 24 hours than did the commercially available docetaxel, Taxotere®.

[0455] Quantification of docetaxel in tissue and plasma samples was performed using liquid chromatography / tandem mass spectrometry (LC-MS) as described in Example 1. Tissue samples were weighed and homogenized in 8 mL of 0.01 M PBS per gram of tissue using a gentleMACS™ Dissociator (Miltenyi Biotec). Drug extraction was performed by protein precipitation using acetonitrile. To do this, 900 microliters of acetonitrile containing 9 mg of the internal standard paclitaxel was added to 100 microliters of plasma or homogenized tissue samples. The mixture was then vortexed for 20 minutes and centrifuged at 20,817 g for 5 minutes. 800 microliters of the resulting supernatant was collected and dried by evaporation (MiVac Duo Concentrator, Genevac) at 40°C. Finally, the resulting dried sample was dissolved in 100 microliters of mobile phase, filtered through a 0.22 micrometer pore size (Millex-GV 4 mm, Millipore), and transferred to an LC vial. Calibration standards were also prepared by spiking blank plasma or tissue samples with the docetaxel standard solution. Under these conditions, the internal standard paclitaxel eluted at 4.17 + / - 0.01 minutes, and the transitions from 854.6 to 286 and 854.6 to 569 were monitored. Data collection and analysis were performed using TargetLynx v4.1 software (Waters).

[0456] In this example, we compared the efficacy of tLyp1-functionalized PSA nanocapsules with that of a commercially available formulation, Abraxane® (paclitaxel), in a PDX (patient-derived transplant) pancreatic cancer mouse model. The results in Figure 3 demonstrate that tLyp1-functionalized PSA nanocapsules (Ratio 2) were more effective than Abraxane®. Tumor growth was significantly reduced, and mouse survival was significantly prolonged (42 days vs. 56 days). Furthermore, the nanocapsules exhibited low in vivo toxicity (as measured by weight loss in healthy mice) (Figure 4) and hematologic toxicity.

[0457] Figure 2 shows the accumulation of docetaxel at 1 hour (Figure 2A) and 24 hours (Figure 2B) after IV administration of an equivalent docetaxel dose of 7.5 mg / kg Taxotere® (commercially available docetaxel), PSA NC, PSA-tLyp1 NC, and tLyp1+PSA NC. Data are presented as the mean + / - standard deviation (SD) of five replicates. Significant differences between treatments ( * )p<0.01.

[0458] Figure 3 shows the relative tumor volumes after IV administration of Abraxane® (paclitaxel dose 150 mg / Kg) and docetaxel-loaded tLyp1-PSA nanocapsules (docetaxel dose 60 mg / kg). All data are shown as the mean + / - standard error of the mean (SEM) of five replicates. Mice died or were sacrificed on day 42 (control and Abraxane®-treated) or day 56 due to progression of the disease state.

[0459] Figure 4 shows the weight change in mice treated with tLyp1-PSA nanocapsules at an equivalent total docetaxel dose of 75 mg / kg. All data are presented as the mean + / - standard deviation (SD) of five replicates.

[0460] Example 3 In addition to tLyp1, other targeting and / or tissue-penetrating peptides, such as CendR peptides (eg, cLyp1 and iRGD), can be attached to polymer chains, such as PSA.

[0461] cLyp1 was covalently conjugated to PSA using a chemical strategy similar to that used for PSA-tLyp1. First, PSA was modified with N-(2-aminoethyl)maleimide trifluoroacetate. Various molar ratios between the carboxylic acid groups of PSA and EDC, NHS, AEM, and the peptide (cLyp1) were tested (Table 4). For this purpose, PSA was dissolved in 0.1 M MES buffer at pH 6 to a final concentration of 2 mg / mL. Corresponding amounts of EDC, NHS, and AEM were also dissolved in 0.1 M MES buffer, added to the PSA solution, and maintained under magnetic stirring at room temperature for 4 hours. Maleimide-functionalized PSA (PSA-Mal) was purified by dialysis first against 50 mM NaCl and then against MilliQ water, as described in Example 1. In the second step, PSA-Mal was dissolved in a solution of 0.1 M MES buffer and 50 mM NaCl to a PSA concentration of 1 mg / mL. To this solution, the linear peptide (H-CC(Acm)GNKRTRGC(Acm)-OH), with acetamidomethyl protecting groups on cysteines 2 and 10 and no protecting group on cysteine ​​1, was added, and the reaction mixture was kept under magnetic stirring at room temperature for 24 hours. The PSA modified with the protected linear peptide was purified by dialysis under the same conditions as above. To obtain the final cyclic peptide, deprotection of cysteines 2 and 10 of the peptide was carried out by adding 1 mL of 1 M HCl to the PSA-peptide solution, followed by the addition of 1 molar equivalent of I2 (5.10 mL in methanol) relative to the peptide. -3 A solution of iodine in methanol (Sigma-Aldrich) containing iodine (I) (I M) was added to the conjugate under magnetic stirring for 1 h to carry out the cysteine ​​oxidation reaction, and then one drop of 1 M ascorbic acid (Panreac) in water was added to the solution to neutralize any excess I2 from the medium. The final PSA-cLyp1 product was purified by dialysis as previously described in Example 1, lyophilized, and stored at 4°C.

[0462] Characterization of PSA-cLyp1 conjugates 1 H-NMR was performed.

[0463] [Table 4]

[0464] Preparation of nanocapsules using PSA-cLyp1 and PSA-tLyp1 by self-emulsification technique. Briefly, 1.75 mL of aqueous phase containing 5.95 mg of PSA-cLyp1 was added to a magnetically stirred organic phase containing 118 mg of Labrafac lipophile WL1349 (Gattefosse), 116 mg of polysorbate 80 (Tween 80, Merck), 5 mg of Macrogol 15 Hydroxystearate (Kolliphor HS15®, BASF), 0.4 mg of benzethonium chloride (Spectrum Chemical), 2 mg of docetaxel anhydrous (Hao Rui Enterprises), and 50 microliters of ethanol.

[0465] The nanocapsules were characterized in terms of mean particle size, polydispersity index (PI), and zeta potential according to the methods in Example 1 above. The total docetaxel content in an aliquot of unisolated nanocapsules was estimated. Drug quantification was performed by UPLC according to the method previously described in Example 1. Results are expressed as the mean + / - SD of three replicates (Table 5).

[0466] [Table 5]

[0467] Preliminary in vivo efficacy studies. The efficacy of cLyp1- and tLyp1-functionalized PSA nanocapsules (5 mg / kg docetaxel) was compared with that of the commercially available formulations Abraxane® (paclitaxel, 15 mg / kg) and Taxotere® (docetaxel, 5 mg / kg) in a metastatic orthotopic lung cancer model (A549 cells) in mice (n = 3–4 per group). Figure 5 shows quantification of ex vivo luciferase activity in (i) the lungs (Figure 5A) and (ii) the mediastinal lymph nodes (Figure 5B) after various treatments (TAXO, taxotere; ABRAX, Abraxane®; A, PSA-tLyp1 nanocapsule ratio 30; B, PSA-cLyp1 ratio 20; C19, untreated control on day 19; C37, untreated control on day 37).

[0468] The results in Figure 5 show that both functionalized formulations had similar responses in terms of tumor cell reduction in the lungs and mediastinal (metastatic) lymph nodes. Interestingly, the functionalized nanocapsules were more effective at suppressing metastasis than Abraxane® and Taxotere®. Furthermore, analysis of weight loss, hematology, and histopathology of vital organs revealed no signs of toxicity with the nanocapsules (data not shown).

[0469] Example 4 This example demonstrates the feasibility of increasing batch production of nanocapsules and establishes a scalable technique. Thus, for example, larger batches of PSA nanocapsules were prepared at a 10-fold scale (110 mL batches) by solvent displacement.

[0470] The organic phase contained 10 mL of ethanol containing 37.5 mg of phosphatidylcholine (Lipoid S100, Lipoid), 7.5 mg of benzethonium chloride (Spectrum Chemical), 152.8 mg of caprylic / capric triglyceride (Labrafac Lipophile WL 1349, Gattefosse), and 7.5 mg of docetaxel anhydrous (Hao Rui Enterprises). The aqueous phase consisted of 100 mL of a 0.25 mg / mL PSA solution. The aqueous phase was maintained using a top-mounted propeller stirrer (Ika RW 20 Digital) with a four-blade propeller (10M / M-P15) at 700 rpm, and the organic phase was pumped into the aqueous phase through a peristaltic pump tubing (1.6 × 4.8 × 1.6 platinum-cured silicone, Freudemberg) using a peristaltic pump (Minipuls 3, Gilson) at 25 rpm. After nanocapsule formation, the organic solvent was removed by rotary evaporation.

[0471] Following isolation / concentration by ultracentrifugation, the nanocapsules were characterized in terms of mean particle size, polydispersity index (PI), and zeta potential according to the methods described above. Drug quantification was performed by UPLC according to the method already described for AE% in Example 1. The results, corresponding to three independent replicates, are shown in Table 6.

[0472] [Table 6]

[0473] Furthermore, isolation / concentration by tangential flow filtration was evaluated as an alternative to ultracentrifugation. Tangential flow filtration is a scalable method that ideally eliminates the rotary evaporation step. Therefore, crossflow tests were performed using a Sartoflow Smart Crossflow System (Sartorius). In these tests, a 1 L volume of docetaxel-loaded PSA nanocapsules (pooled from 10 individual batches of 110 mL each without rotary evaporation) was successfully concentrated at least 20-fold in a cassette Hydrosart 100 kDa. The mean flow rate (LMH) was 120.6 L / hm. 2 The isolation time, enrichment factor, final docetaxel concentration, and docetaxel binding efficiency (indirect AE % measured in the filtrate) for three replicates are shown in Table 7.

[0474] [Table 7]

[0475] Scaling of batch production was also evaluated using self-emulsifying technology (100 mL batch size) by adding an aqueous phase containing 297.5 g of polymer and 87.5 g of water to an organic phase containing 5900 mg of Labrafac Lipophile WL1349 (Gattefosse), 5800 mg of Polysorbate 80 (Tween 80, Merck), 250 mg of Macrogol 15 Hydroxystearate (Kolliphor HS15®, BASF), 20 mg of benzethonium chloride (Spectrum Chemical), 100 mg of docetaxel anhydrous (Hao Rui Enterprises), and 500 μL of ethanol using a top-mounted propeller agitator (IKA RW 20 Digital) with a four-blade propeller (10M / M-P15) at 1000 rpm.

[0476] The nanocapsules were characterized in terms of mean particle size, polydispersity index (PI), and zeta potential according to the method in Example 1 above. The total docetaxel content / concentration in an aliquot of non-isolated nanocapsules was estimated. Drug quantification was performed by UPLC according to the method previously described in Example 1. The results corresponding to three replicates (PSA nanocapsules) and one replicate (PSA-tLyp1 NC ratio 10 and ratio 20) are shown in Table 8.

[0477] [Table 8]

[0478] Preliminary studies on the isolation / concentration of one pool of 10 100 mL batches by tangential flow filtration (Sartoflow Smart Crossflow System, Sartorius) were carried out according to the conditions previously described. The total docetaxel content / concentration in an aliquot of isolated nanocapsules (retentate) was estimated, whereas the AE% was determined in two different ways: (i) directly (retentate analysis) and (ii) indirectly (filtrate analysis). Drug quantification was performed by UPLC according to the method previously described in Example 1. The results are shown in Table 9.

[0479] [Table 9]

[0480] Example 5 This example describes the preparation of PSA nanocapsules conjugated with other lipophilic small molecules, such as the anticancer drugs paclitaxel (856.903 g / mol; Log P 3.2; Teva) and patupilone (507.686 g / mol; Log P 3.7; Sigma-Aldrich).

[0481] Paclitaxel-loaded PSA nanocapsules were prepared using a Nanoassemblr® Benchtop microfluidics device (Precision Nanosystems) as follows: The aqueous phase consisted of 10 mL of a 0.25 mg / mL PSA solution. The organic phase consisted of 1 mL of ethanol containing 3.75 mg of Lipoid S100 (Lipoid), 0.75 mg of benzethonium chloride (Spectrum Chemical), 15.3 mg of Labrafac Lipophile WL 1349 (Gattefosse), and 0.75 mg of paclitaxel (Teva). Briefly, both the aqueous and organic phases were injected into each inlet of the NanoAssemblr cartridge at a total flow rate of 8 mL / min. After nanocapsule formation, the ethanol was removed by rotary evaporation.

[0482] Patupilone-loaded PSA nanocapsules were also prepared using the above conditions, substituting only paclitaxel with 0.75 mg of patupilone, using a Nanoassemblr® Benchtop microfluidics device (Precision Nanosystems).

[0483] The nanocapsules were characterized in terms of mean particle size, polydispersity index (PI), and zeta potential after isolation / concentration by ultracentrifugation according to the method described above in Example 1. Quantification of drug for AE% was performed by two different analytical methods, briefly: (i) Paclitaxel: Drug quantification was performed by UPLC. The UPLC system included an Acquity UPLC® H-Class System (Waters) and a column compartment (BEH C18 column, 2.1 × 100 mm, 1.7 micrometers, Waters). The experimental analysis conditions were as follows: the mobile phase included MilliQ water (A) and acetonitrile (B). An isocratic program of 55% A and 45% B was used. The flow rate was 0.6 mL / min, and the run time was 4 minutes. The column temperature was maintained at 40°C, and the autosampler was thermostated at 4°C. The injection volume was 10 microliters. Under these conditions, paclitaxel eluted at 1.3 minutes. (ii) Patupilone: ​​Drug quantification was performed by HPLC. The HPLC system included a VWR Hitachi ELITE LaChrom (Hitachi) and a column compartment ACE Equivalence reversed-phase C18 (5 micrometers × 250 mm × 4.6 mm). The experimental analysis conditions were as follows: the mobile phase included MilliQ water (A) acidified with 0.1% formic acid and acetonitrile (B). An isocratic program of 80% A and 20% B was used. The flow rate was 1 ml / min, and the run time was 7.0 min. The column temperature was maintained at 30°C. The injection volume was 50 microliters. The detection wavelength was set at 248 nm. Under these conditions, patupilone eluted at 3.55 min.

[0484] The results corresponding to three replicates for both paclitaxel and patupilone formulations are shown in Table 10.

[0485] [Table 10]

[0486] Example 6 C 12 One example for the preparation of functionalized PSAs is shown below. 12(dodecyl) is used, but other alkyl groups may be used in other experiments as well. Referring to Figure 5, PSA sodium salt (30 kDa) was treated with Dowex, followed by tetrabutylammonium hydroxide. After concentration / purification by ultrafiltration and lyophilization of the concentrate, the tetrabutylammonium salt of PSA, which is readily soluble in DMF, was obtained.

[0487] The acid was then activated with 2-bromo-1-ethylpyridinium tetrafluoroborate and then reacted with dodecylamine. After isolation of the product by precipitation, the tetrabutylammonium cation was replaced with sodium cation. Concentration and purification by ultrafiltration and lyophilization of the concentrate afforded the desired dodecylamide-functionalized PSA sodium salt. 1 H-NMR analysis confirmed the structure and the degree of substitution in the range of 4%.

[0488] Several test reactions were performed to further optimize the amount of 2-bromo-1-ethylpyridinium tetrafluoroborate. Initial tests using 5% 2-bromo-1-ethylpyridinium tetrafluoroborate resulted in very low incorporation of dodecylamine into the polymer (less than 1%). Experiments using 1 equivalent of 2-bromo-1-ethylpyridinium tetrafluoroborate yielded a product that was poorly soluble in water. Using 30% 2-bromo-1-ethylpyridinium tetrafluoroborate, degrees of substitution in the 4% range were obtained. The reaction was then scaled up to produce 1 gram of functionalized polymer.

[0489] Ultrafiltration. PSA tetrabutylammonium salt and derivatized PSA were concentrated (and desalted) using ultrafiltration. Cassette (Pall) 10K Omega centramate T series 0.019 ml 2Ultrafiltration was performed using a Pall Minim II Tangential Flow Filtration (TFF) System using (Part Number OS010T02, Serial Number 36049076R, Membrane Lot Number H5257E). Diafiltration involves continuously feeding water into a reservoir and allowing the permeate to exit. Salts and low molecular weight impurities permeate the membrane and are therefore removed from the PSA solution.

[0490] PSA tetrabutylammonium salt (2). Dissolve PSA sodium salt (1 g) in purified water (100 mL) and add it to Dowex 50WX8 (200-400, H + The resin was stirred with 20 mL of methylcellulose (freshly washed with water, then methanol, then water) for 30 minutes, filtered off, and washed with deionized water. The pH of the solution was less than 4. The solution was treated with tetrabutylammonium hydroxide (40 wt. % aqueous solution) until the pH was approximately 12. This entire procedure was repeated twice, and then the final pH was adjusted to 7.5-8 by bubbling CO2 and then N2.

[0491] Ultrafiltration. The solution of PSA tetrabutylammonium salt was placed in a reservoir (400 mL) and concentrated to a volume of 100 mL. During diafiltration, water was continuously fed into the reservoir (300 mL). The permeate flow rate was 12 mL / min. At the end of diafiltration, the solution was further concentrated to a minimum volume and removed from the reservoir. The transmembrane pressure during diafiltration was 0.6 bar, P1 = 1.2 bar. The concentrate was freeze-dried to give the title compound (1.6 g) as a white solid.

[0492] Dodecylamide-functionalized PSA tetrabutylammonium salt (3). To a solution of PSA tetrabutylammonium salt 2 (1.3 g, 2.43 mmol / eq) in DMF (30 mL) was added a solution of 2-bromo-1-ethylpyridinium tetrafluoroborate (233 mg, 0.85 mmol, 0.35 eq) in DMF (1 mL) at room temperature under N2, and the solution was stirred for 1 h. A solution of 1-aminododecane (270 mg, 1.46 mmol) and Et3N (0.576 mL, 4.13 mmol) in DMF (1 mL) was added to the reaction, and the mixture was stirred for 40 h. The reaction mixture was added dropwise to a solution of Et2O (150 mL) and acetone (15 mL). The precipitate was collected by filtration, washed with Et2O, and dried under reduced pressure.

[0493] Dodecylamide-functionalized PSA sodium salt. Dissolve the white precipitate in deionized water (100 mL) and wash the solution with Dowex 50WX8 (200-400, H + The resin was stirred with 20 mL of HCl (freshly washed with water, then methanol, then water) for 30 min, filtered off, and washed with deionized water. The pH of the solution was less than 4. The solution was treated with aqueous sodium hydroxide (1 M) until the pH was 12. This entire procedure was repeated twice, and then the final pH was adjusted to 7.5-8 by bubbling CO2 and then N2.

[0494] Ultrafiltration. The solution of derivatized PSA sodium salt was placed in a reservoir (500 mL) and concentrated to a volume of 100 mL. During diafiltration, water was continuously fed into the reservoir (500 mL). The permeate flow rate was 10.4 mL / min. At the end of diafiltration, the solution was further concentrated to a minimum volume and removed from the reservoir. The transmembrane pressure during diafiltration was 0.6-0.7 bar (P1 = 1.2-1.3 bar).

[0495] The concentrate was lyophilized to give dodecylamide-functionalized PSA sodium salt 4 (800 mg) as a white solid. 1 H-NMR revealed that the degree of substitution was about 4%.

[0496] Example 7 The bifunctionalized polymer was prepared as follows, although other alkyl groups and targeting peptides may be used in other experiments. To prepare C16-HA-tLyp-1, commercially available C16-HA (MW 55 kDa, SD 7%, Contipro) was used as the starting material, and tLyp1 was chemically conjugated to the carboxylate groups of the HA backbone. The molar ratio of EDC:NHS:AEM:tLyp1 to the carboxylate groups of HA was 1:2.16:0.36:0.072:0.0326 (ratio 4). First, C16-HA was modified with N-(2-aminoethyl)maleimide trifluoroacetate. For this purpose, C16-HA was dissolved in 0.1 M MES buffer at pH 6 to a final concentration of 2 mg / mL. Corresponding amounts of EDC, NHS, and AEM were also dissolved in 0.1 M MES buffer, added to the C16-HA solution, and maintained at room temperature for 4 hours under magnetic stirring. The resulting product was purified by dialysis as described for PSA-tLyp1 in Example 1. In the second step, C16-HA-Mal was dissolved in a solution of 0.1 M MES buffer and 50 mM NaCl at a concentration of 1 mg / mL. tLyp1 was then added to this solution, and the reaction mixture was maintained at room temperature for 24 hours under magnetic stirring. The final product was purified by dialysis as previously described and lyophilized.

[0497] The characterization of this conjugate 1 H-NMR was performed.

[0498] Example 8 This example describes the formulation of various polymeric nanocapsules for efficient conjugation and delivery of monoclonal antibodies (mAbs). As a non-limiting example, the polymeric shell can be made from biodegradable polyacids or polyamides, which can be further functionalized with targeting and / or tumor / tissue-penetrating ligands, such as tLyp-1. PSA (8 kDa, 30 kDa, or 94 kDa, Serum Institute of India), or PSA-tLyp1 ratio 20, or C12-PSA (Example 6), or HA (330 kDa, Lehvoss Iberica), or C16-HA (various Mw and alkyl substitution: 55 kDa - SD 7%; 216 kDa - SD 5%; 216 kDa - SD 11%, Contipro), or C16-HA-tLyp1 (Example 7), or polyglutamic acid (PGA, 11.9 kDa, Polypeptide Therapeutic Solutions), or PGA-PEG (PGA 6.68 kDa / PEG 5 kDa, Polypeptide Therapeutic Solutions), or polyamide polyaspartic acid (PASP, poly-L-aspartic acid, 200 units, average Mw Nanocapsules with polymer coatings of PEG (Methoxy-poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt, mPEG5K-b-PLD200, average MW 32 kDa, Alamanda Polymers) or PASP-PEG (Methoxy-poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt, mPEG5K-b-PLD200, average MW 32 kDa, Alamanda Polymers) were prepared by a self-emulsification technique.

[0499] Preparation of blank nanocapsules (without antibody). First, 59 mg of polysorbate 80 (Tween 80®, Merck) and 58 mg of caprylic / capric triglyceride (Mygliol® 812N, IOI Oleochemical) were weighed into a 2 mL glass vial (oil phase). Next, for formulations containing non-hydrophobically modified or non-amphiphilic polymers as the shell, a cationic surfactant was added to the oil phase (4 microliters of benzethonium, 50 mg / mL, previously solubilized in ethanol). All components of the oil phase were maintained under magnetic stirring (500 rpm). In parallel, aqueous phases were prepared by separately solubilizing each polymer at various concentrations (e.g., 3 mg / mL for PSA-based formulations, 0.25 mg / mL for HA-based formulations, 3 mg / mL for PGA, 6 mg / mL for PGA-PEG, 3 mg / mL for PASP, and 6 mg / mL for PASP-PEG) in 25 mM PBS, pH 7.3. Macrogol 15 Hydroxystearate (Kolliphor HS15®, BASF) was also solubilized at a concentration of 20 mg / mL in 25 mM PBS, pH 7.3. Then, 0.75 mL of the polymer solution was mixed with 125 microliters of Kolliphor solution, and the resulting aqueous phase was added to the oil phase under magnetic stirring (1100 rpm).

[0500] Binding of mAbs. Two different methods were used: (i) One-step method: The required volume of mAb solution was added to the aqueous phase to obtain the desired final mAb concentration (e.g., 0.5 mg / mL), and then mixed with the oil phase. The mAbs conjugated to the nanocapsules in the one-step method were anti-PD-L1 mAb (rat anti-mouse IgG2a, BioXcell®) and bevacizumab (humanized IgG1, Selleck Chemicals). (ii) Two-step method: A solution containing a mAb at a desired concentration (e.g., 1 mg / mL) was added to pre-formed nanocapsules under rotary agitation (550 rpm) to achieve a final mAb concentration of, e.g., 0.5 mg / mL. The mAb was incubated with the nanocapsules at room temperature for 4 hours. A non-limiting example of a mAb that can be conjugated to nanocapsules using the two-step method is anti-PD-L1 mAb (rat anti-mouse IgG2a, BioXcell®) (Table 14).

[0501] The nanocapsules were characterized in terms of mean particle size, polydispersity index (PI), and zeta potential according to the methods described above. The results corresponding to three replicates are shown in Table 13 (one-stage method, anti-PD-L1), Table 14 (two-stage method, anti-PD-L1), and Table 16 (one-stage method, bevacizumab).

[0502] Monoclonal antibody binding efficiency. To determine the binding of mAb to the nanocapsules, 1 mL aliquots of each different formulation were filtered through Amicon Stirred Cells® (polyethersulfone Biomax® 500 KDa Ultrafiltration Discs, Merck) under 1 bar of nitrogen pressure at 4°C. After this isolation step, the filtrate containing free mAb was collected and analyzed by the corresponding ELISA assay. Binding efficiency was indirectly calculated as (total mAb - free mAb) / total mAb × 100. The results are shown in Table 11 (one-step method, anti-PD-L1), Table 14 (two-step method, anti-PD-L1), and Table 16 (one-step method, bevacizumab).

[0503] Leakage upon dilution at room temperature (RT). To assess whether mAbs are tightly encapsulated within the nanostructures, mAb binding was assessed upon dilution (1:2->1:16) with PBS (25 mM), pH 7.3 at RT, following the method described above for mAb binding efficiency. The results obtained for the different mAb binding methods are shown in Tables 12 and 16 for the one-step formulation and in Table 15 for the two-step formulation.

[0504] In vitro release study. mAb-loaded nanocapsules were incubated (1:10 dilution) in PBS (25 mM), pH 7.3 at 37°C. At predetermined times (1 hour and 2 hours), samples were collected and filtered according to the method described above, and the released mAb was quantified by ELISA (Table 13, one-step method, anti-PD-L1).

[0505] Tables 11-16 show that it is possible to formulate various polymer nanocapsules that exhibit suitable physicochemical properties and high conjugation efficiency for various mAbs. The conjugation of mAbs can be performed, for example, by the one-step and two-step methods described above, although the one-step method provides better encapsulation of mAbs within the nanostructures, as can be inferred from the performed mAb leakage studies upon dilution (Table 12 for one-step; Table 15 for two-step).

[0506] [Table 11]

[0507] [Table 12]

[0508] [Table 13]

[0509] [Table 14]

[0510] [Table 15]

[0511] [Table 16]

[0512] Cytotoxicity of various empty blank polymer nanocapsules (without mAb). Cytotoxicity was determined using a crystal violet assay as an indicator of cell viability. MDA-MB-231 cells seeded in 96-well tissue culture plates were co-incubated with the above-dispersed formulations (at various concentrations) in cell culture medium for 2 hours, after which cell viability was assessed. As can be seen in Figure 7, in all cases, cell viability was greater than 80% at concentrations up to 6 mg / mL.

[0513] Morphological analysis of mAb-loaded polymer nanocapsules. Morphological analysis of mAb (bevacizumab)-loaded nanocapsules was performed using a transmission electron microscope (TEM, CM12, Philips, The Netherlands). For TEM observation, samples were stained with phosphotungstic acid (2%, w / v) solution and placed on a copper grid coated with Formvard®. TEM images of PSA nanocapsules (A) and HA 216 SD5% (B) containing bevacizumab (final concentration 3 mg / mL) are shown in Figure 8 (size bars: 1 micrometer for Figures 8A and 8C, 200 nm for Figures 8B and 8D).

[0514] Freeze-drying study. Furthermore, freeze-drying studies were conducted to evaluate whether the mAb-containing nanocapsule suspension could be processed into a powder for long-term storage. As a non-limiting example, various bevacizumab-loaded polymer nanocapsules were prepared using the one-step method described above. A concentrated solution of trehalose and mannitol was added to the nanocapsule suspension (final concentrations of trehalose 5% w / v and mannitol 2.5% w / v), followed by freeze-drying (approximately 50-hour cycles; Pilot Lyophilizer VirTis Genesys 25 ES). The stability of the freeze-dried nanocapsules stored at 4°C for 4 months was analyzed by measuring particle size, PI, pH, zeta potential, and total mAb content (by ELISA) and comparing them with the initial (pre-lyophilization) values. Measurements were performed using the same methods as described above. The results, corresponding to three replicates, are shown in Table 17, which indicates that after 4 months of storage, there were no significant changes in the physicochemical properties, while the total mAb percentage was approximately 80–90%.

[0515] [Table 17]

[0516] Example 9 This example describes the formulation of alternative polymeric nanocapsules for efficient conjugation and delivery of monoclonal antibodies (mAbs). The polymeric shell can be made of biodegradable, water-insoluble polymers such as PEGylated poly(lactic-co-glycolic acid) (PLGA-PEG or PLG-PEG) or PEGylated polylactic acid (PLA-PEG), which can be further functionalized with targeting and / or tumor / tissue-penetrating ligands, such as tLyp-1.

[0517] Nanocapsules with a polymer coating of PLA-PEG were prepared by the solvent displacement method and antibody was conjugated by a one-step method. Briefly, the preparation of a 5 mL batch was as follows: (1) Preparation of oil phase: 290 mg of polysorbate 80 (Tween 80®, Merck) and 295 mg of Mygliol® 812N (IOI Oleochemical) were weighed into a 25 mL glass vial and mixed under magnetic stirring (500 rpm). PLA-PEG polymer was solubilized with 12.5 mL of acetone, added to the previous solution, and maintained under magnetic stirring (500 rpm); (2) Preparation of aqueous phase: 625 microliters of a solution of Kolliphor HS15® (20 mg / mL in 25 mM PBS, pH 7.3) was mixed in a 100 mL glass vial with 4.4 mL of 25 mM PBS, pH 7.3 containing the corresponding amount of mAb and 25 mL of water.

[0518] The oil phase was then added to the aqueous phase under magnetic stirring (1250 rpm) using a 20 mL syringe (120 × 40 mm needle). Nanodroplets immediately formed, with the polymer surrounding them. The final NC suspension was rotary evaporated until it reached 5 mL. Following the methods described above, the nanocapsules were characterized in terms of mean particle size, polydispersity index (PI), zeta potential, and binding at a 1:16 dilution. Results corresponding to three replicates are shown in Table 18.

[0519] [Table 18]

[0520] Example 10 One of the major limitations of nanocarriers is their low drug binding efficiency and loading capacity at clinically translatable doses. Therefore, we evaluated the effect of antibody concentration on the physicochemical properties of PSA nanocapsules, as well as their mAb binding efficiency and encapsulation, using bevacizumab (Selleck Chemicals) as a mAb model. A one-step method was used for mAb binding.

[0521] Following the methods described above (Example 8), the nanocapsules were characterized in terms of mean particle size, polydispersity index (PI), zeta potential, binding efficiency, and binding at a 1:16 dilution. The results, corresponding to three replicates, are shown in Table 19.

[0522] A final bevacizumab concentration of at least 5 mg / mL was achieved without significantly affecting the nanocapsule properties and while maintaining a high conjugation efficiency of 70%, which corresponds to a mAb loading content of approximately 3% (mAb loading content = weight of conjugated mAb / total weight of nanocapsule components).

[0523] [Table 19]

[0524] Example 11 In many cases, the ineffectiveness of nanocarriers is the result of their aggregation in complex media, which may be due to the high ionic strength of the biological medium and / or the presence of proteins. Therefore, we investigated the stability of various mAb-loaded polymer nanocapsules in plasma as an indicator of the potential for parenteral administration of mAbs.

[0525] Stability in Plasma. Bevacizumab-loaded nanocapsules prepared by the one-step method of Example 8 were incubated in mouse plasma (1:10 dilution, 37°C) under horizontal shaking (300 rpm, Heidolph Instruments). At designated times, samples of the incubation environment were taken and subjected to particle size analysis using a Malvern Zeta-Sizer and size and size distribution analysis using Nanoparticle Tracking Analysis (NTA). Samples were further diluted as appropriate (1:10,000 in 10 mM PBS, pH 7.4 for NTA; 1:1,000 in water for DLS) before analysis.

[0526] The stability of various mAb-loaded polymer nanocapsules as measured by DLS is shown in Figure 9 (Figure 9A: C16-HA-based nanocapsules; Figure 9B: PSA-based nanocapsules). Results are the average of three replicates.

[0527] The stability of various mAb-loaded polymer nanocapsules as measured by NTA is shown in Figure 10 (Figure 10A: C16-HA-based nanocapsules; Figure 10B: PSA-based nanocapsules; Figure 10C: PGA-based and PLA nanocapsules; n=1).

[0528] Both mAb-loaded nanocapsules exhibited sufficient stability in complex media such as plasma for at least 24 hours, which is an important advantage for parenteral administration to subjects.

[0529] Example 12 In this example, we demonstrate that various polymer nanocapsules can interact with cells in vitro and induce cellular internalization of conjugated antibodies. To carry out this study, various nanocapsules conjugated with a fluorescent antibody model (FITC-IgG, purity >98%, Elabsciences) were prepared by a one-step method as described in Example 8 and characterized in terms of size, PI, and zeta potential (Table 20).

[0530] [Table 20]

[0531] First, we performed flow cytometry experiments to determine whether the nanocapsules could interact with cells. Various polymer nanocapsules (diluted in cell culture medium to a final concentration of 7 mg / mL nanocapsules and 105 micrograms / mL IgG-FITC) were added to cultured MBD-MB-231 cells (66,500 cells / well) and incubated for 2 hours at 37°C (37°C, 5% CO2, humidified incubator). After incubation, the cells were gently washed twice with PBS and then trypsinized for flow cytometry analysis. Figure 11 shows the percentage of positive cells after 2 hours of incubation with various polymer nanocapsules for three replicates. The percentage of positive cells was approximately 40–55% for all FITC-IgG-loaded nanocapsules, demonstrating the excellent ability of the nanocapsules to interact with cells within a short period of time (2 hours).

[0532] Further testing was then performed using a more advanced imaging flow cytometer (ImageStream®) to determine whether the nanocapsules could induce effective cellular internalization of the bound antibody. Briefly, FITC-IgG-loaded nanocapsules were incubated with A549 cells (1 mL of DMEM containing 6 mg / mL nanocapsules per well) in a 6-well plate, using separate wells for each time point to be tested (e.g., 0 min, 30 min, 2 h, 4 h, 6 h, and 24 h). At each given time point, cells were trypsinized and images were acquired using the ImageStream® instrument to determine the percentage of nanocapsule-positive cells (Figure 12A) and the corresponding FITC-IgG internalization score (Figure 12B). Effective internalization was determined by labeling cytoplasmic acidic organelles with Lysotracker®, a fluorescent marker for live cells, and further confirmed by confocal microscopy (data not shown).

[0533] As seen in Figure 12 , FITC-IgG-loaded PSA, PSA-tLyp1, and C16-HA216 SD5% nanocapsules induced effective translocation of the bound antibody model into the cell interior in a time-dependent manner up to 100% positive cells.

[0534] Thus, this example demonstrates that polymer nanocapsules may facilitate cellular internalization of conjugated mAbs.

[0535] Example 13 This example demonstrates the feasibility of combining two active substances with very different properties and sizes within the same nanocapsule. As a non-limiting example, we formulated C16-HA 216 SD5%, C16-HA 55 SD7%-tlyp nanocapsules, and PSA nanocapsules using the self-emulsifying technique with both the mAb bevacizumab (a water-soluble polymer) and paclitaxel (a lipid-soluble small molecule).

[0536] Briefly, the oil phase was prepared by weighing 290 mg of polysorbate 80 (Tween 80®, Merck), 295 mg of caprylic / capric triglyceride (Labrafac Lipophile WL 1349®, Gattefose), 12.5 mg of Kolliphor HS15 (BASF), and 5 mg of paclitaxel into a glass vial and stirring all components under magnetic stirring at 700 rpm to thoroughly mix and solubilize them. For PSA nanocapsules, the oil phase additionally contained benzethonium chloride, as previously reported for non-amphiphilic polymers in Example 8.

[0537] In parallel, the polymers were separately solubilized in PBS (25 mM) at pH 7.3 (0.25 mg / mL for C16-HA nanocapsules and 3 mg / mL for PSA nanocapsules), and the aqueous phase was prepared by adding the corresponding amount of bevacizumab to a final formulation concentration of 0.5 mg / mL. 4.415 mL of the aqueous phase was then added to the oil phase (597.5 mg) under magnetic stirring (1250 rpm, 10 min).

[0538] Drug quantification was performed by HPLC. The HPLC system included a VWR Hitachi ELITE LaChrom (Hitachi, Tokyo, Japan) and a column compartment ACE Equivalence reversed-phase C-18 (5 micrometers × 250 mm × 4.6 mm; Aberdeen, Scotland). The experimental analysis conditions were as follows: the mobile phase included MilliQ water (A) and acetonitrile (B). An isocratic program of 40% A and 60% B acidified with 0.1% trifluoroacetic acid was used. The flow rate was 1.5 ml / min, and the run time was 10.0 min. The column temperature was maintained at 30°C, the injection volume was 25 microliters, and the UV detector was at 227 nm. Under these conditions, PCX eluted at 4.21 + / - 0.02 min.

[0539] Measurements of size, PDI, zeta potential, and bound mAb were performed using the same methods as previously described for mAb-loaded nanocapsules (Example 8). The total amounts of mAb and palitaxel in the unisolated nanocapsule samples were analyzed by the corresponding ELISA and HLPC methods, respectively. The results are shown in Table 21.

[0540] [Table 21]

[0541] Reference list Curr.Org.Chem.,17(9):975-998,2013. Giorgi, et al., “Carbohydrate PEGylation, an approach to improve pharmacological potency,” Beilstein J.Org.Chem., 10:1433-44, 2014. Bertrand N., et al., Cancer Nanotechnology: The impact of passive and active targeting in the era of modern cancer biology, Advanced Drug Delivery Reviews 66(2014)2-25. Gilad Y., et al., Recent innovations in peptide based targeted delivery to cancer cells, Biomedicines, 4(2016). Zhou G.,et al.Aptamers:A promising chemical antibody for cancer therapy,Oncotarget,7(2016)13446-13463. Zhang D.et al.,Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems,Journal of Controlled Release,Volume 229(2016)Pages 130-139. Regberg J.,et al.Applications of cell-penetrating peptides for tumor targeting and future cancer therapies,Pharmaceuticals,5(2012)991-1007. Ruoslahti E.,Tumor penetrating peptides for improved drug delivery,Advanced Drug Delivery Reviews,Volumes 110-111(2017)Pages 3-12. Mojarradi,“Coupling of substances containing a primary amine to hyaluronan via carbodiimide-mediated amidation,”Master’s Thesis,Uppsala University,March,2011.

Claims

1. 1. A composition comprising a plurality of nanocapsules comprising an inner portion surrounded by an outer shell, said outer shell comprising a polymer and a targeting moiety, said inner portion comprising at least one hydrophobic compound, The polymer may be polysialic acid (PSA) and / or pegylated-polysialic acid (PSA-PEG), hyaluronic acid (HA) and / or pegylated-hyaluronic acid (HA-PEG), Polyglutamic acid (PGA) and / or pegylated-polyglutamic acid (PGA-PEG), Poly(aspartic acid) (PASP) and / or PEGylated-poly(aspartic acid) (PASP-PEG), Polyasparagine and / or pegylated-polyasparagine, alginate and / or pegylated alginate, selected from the group consisting of polymalic acid and / or PEGylated polymalic acid, and mixtures thereof; the hydrophobic compound is an oil, the targeting moiety is selected from the group consisting of Lyp1, tLyp1, cLyp1, iNGR, iRGD, RPARPAR, TT1, linear TT1, RGD-4C, cRGD, cilengitide, F3, 9-RGD, RGD4C, delta24-RGD, delta24-RGD4C, RGD-K5, acyclic RGD4C, bicyclic RGD4C, c(RGDfK), c(RGDyK), E-[c(RGDfK)2], E[c(RGDyK)]2, KLWVLPKGGGC, CDCRGDCFC, LABL, angiopeptin-2, an antibody, a nanobody, transferrin, ankyrin repeat protein, an affibody, folate, triphenylphosphonium, ACUPA, PSMA, a carbohydrate moiety, and an aptamer. composition.

2. The polymer may be polysialic acid (PSA) and / or pegylated-polysialic acid (PSA-PEG), hyaluronic acid (HA) and / or pegylated-hyaluronic acid (HA-PEG), Polyglutamic acid (PGA) and / or pegylated-polyglutamic acid (PGA-PEG), Poly(aspartic acid) (PASP) and / or PEGylated-poly(aspartic acid) (PASP-PEG), and mixtures thereof. The composition of claim 1.

3. 2. The composition of claim 1, wherein the targeting moiety is selected from the group consisting of Lyp1, tLyp1, cLyp1, iNGR, iRGD, RPARPAR, TT1, linear TT1, and F3.

4. The composition of claim 3 , wherein the targeting moiety comprises Lyp1, tLyp, or cLyp1.

5. The composition of any one of claims 1 to 4, wherein the targeting moiety is electrostatically bound to the polymer.

6. The composition of any one of claims 1 to 4, wherein the targeting moiety is attached to the polymer via a linker.

7. The composition of any one of claims 1 to 6, wherein at least a portion of the polymer is associated with a hydrophobic moiety.

8. 8. The composition of claim 7, wherein the hydrophobic moiety is selected from alkyl groups, cycloalkanes, bile salts and derivatives, terpenoids, terpenes, terpene-derived moieties, and fat-soluble vitamins.

9. The composition of claim 8, wherein the hydrophobic moiety comprises a C2-C24 straight chain alkyl group.

10. 10. The composition of claim 9, wherein the hydrophobic moiety comprises a straight chain C16 alkyl group or a C12 alkyl group.

11. The composition of any one of claims 1 to 10, wherein at least about 90% by weight of the shell comprises a polymer.

12. The composition of any one of claims 1 to 11, wherein at least a portion of the plurality of nanocapsules are nanocapsules having an average diameter of less than 1 micrometer.

13. The composition of any one of claims 1 to 12, wherein at least a portion of the plurality of nanocapsules further comprises one or more surfactants.

14. The composition of any one of claims 1 to 13, wherein the polymer is polysialic acid.

15. The targeting moiety is an aminoalkyl (C １ -C ４ ) maleimide linker, aminoalkyl (C １ -C ４ 15. The composition of claim 14, wherein the polysialic acid is linked to the polysialic acid via a methacrylamide linker or directly through an amide group.

16. The aminoalkyl (C １ -C ４ 16. The composition of claim 15, wherein the maleimide linker is generated by an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) or DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) coupling reaction.

17. 17. The composition of claim 15 or claim 16, wherein the targeting moiety is attached to the polysialic acid via an aminoethylmaleimide linker.

18. The targeting moiety is an aminoalkyl (C １ -C ４ ) succinimide linker, aminoalkyl (C １ -C ４ 15. The composition of claim 14, wherein the polysialic acid is linked to the polysialic acid via an amido-iso-propyl linker or directly through an amido group.

19. The aminoalkyl (C １ -C ４ 20. The composition of claim 18, wherein the succinimide linker is generated by an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) coupling reaction.

20. 20. The composition of claim 18 or claim 19, wherein the targeting moiety is attached to the polysialic acid via an aminoethylsuccinimide linker.

21. The composition of any one of claims 1 to 13, wherein the polymer is hyaluronic acid, and at least a portion of the hyaluronic acid is bound to a hydrophobic moiety.

22. 14. The composition of any one of claims 1 to 13, wherein the polymer is PGA and / or PASP and the targeting moiety is attached to the PGA and / or PASP via an aminoalkyl(C1-C4)maleimide linker, an aminoalkyl(C1-C4)methacrylamide linker, or directly through an amide group.

23. 23. The composition of claim 22, wherein the targeting moiety is attached to the PGA and / or PASP via an aminoethylmaleimide linker.

24. The composition of any one of claims 1 to 23, wherein the nanocapsules contain a pharmaceutical agent.

25. 25. The composition of claim 24, wherein the pharmaceutical agent is a monoclonal antibody or a functional fragment thereof.

26. 25. The composition of claim 24, wherein the pharmaceutical agent is an anti-cancer agent.

27. A composition according to any one of claims 1 to 26 for use as a medicament.

28. The composition according to any one of claims 1 to 26, for use in the treatment of cancer.

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