TW201039844A - Biodegradable metal-chelating polymers and vaccines - Google Patents

Abstract

The present invention provides a composition comprising at least one polymer or a salt thereof selected from: a PEA polymer having a chemical formula described by general structural formula (I), or a PEA polymer having a chemical formula described by structural formula (IV):. The use of the composition is also provided.

Description

201039844 VI. INSTRUCTIONS: [Prior Art] Guamino-picoic acid is commonly used as a dissociating agent or integrator in sweat removal in living organisms, and has recently been proposed as a substitute for phosphate in detergents. Already: These compounds form complexes with various metal ions, most commonly with trivalent lanthanides. Polyaminocarboxylic acids such as EDTA (ethylenediaminetetraacetic acid) and DTPA (diethylhexamine-pentaacetic acid) are also commonly used to sequester diagnostic and therapeutic components with in vivo delivery compositions. Polymers with mismatched properties have also been produced. Macromolecular chaos (6) has been reported as a clinical application of MRI contrast agents. For example, ruthenium (1 chelates have been conjugated to biomedical polymers including linear poly(amino acids), polysaccharides, proteins, and various dendritic poly[n]. Copolymerization of DTpA anhydride with diamine has also been reported. And the mismatch with Gd(III). However, the clinical application of such macromolecular systems, including those prepared from typical biodegradable polymers such as dextran, polylysine and the like Constrained by the slow discharge of Gd[ni] complexes and the long-term tissue accumulation of toxic Gd ions. Therefore, despite the advantages of this technology, there is a need for more and better biodegradable avoidance of slow discharge problems. Sub-system. SUMMARY OF THE INVENTION The present invention provides a composition comprising at least one polymer selected from the group consisting of: or a salt thereof: a PEA polymer having a chemical formula of the formula (I), Ο ο Η Ο η μ Ί --c-R1-c-nh-cc-〇-r4.0_^_^_n . l R3 R3 , ^ n Formula (i) 14〇3〇9.d〇c 201039844 where η is about 15 Up to about 150; R1 is independently derived from -CH2-N(CH2C02H)-R6-N(CH2C02H)-CH2- or formula (II) and a combination; wherein R6 is independently selected from the group consisting of (C2-C12)alkyl, p-C6H4, (crc4)alkoxy (c2-c4)alkyl and ch2ch2n(ch2co2h)ch2ch2; and wherein formula (II) The R7 is selected from the group consisting of hydrogen, (C-C12) alkyl and a protecting group;

R3 in the individual η units is independently selected from the group consisting of hydrogen, (CVC6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C10) aryl (CVC6) alkyl, -(CH2)2SCH3, CH2〇H, CH(OH)CH3, (CH2)4NH3+, (CH2)3NHC(=NH2+)NH2, 4-ylidene imidazolinium, CH2COCT, (CH2)2COO_ and combinations thereof a group consisting of; R4 is independently selected from (C2-C20) alkylene, (C2-C20) alkenyl, (C2-C6) alkoxy (C2-C12) alkyl, CH2CH(OH)CH2, CH2CH a group consisting of (CH2OH), a bicyclic ring of a structural formula (III), a 4:3,6-didehydrohexitol, a fragment of 1,4-anhydroerythritol, and a combination thereof;

Formula (III) or a PEA polymer having the formula of formula (IV): 140309.doc 201039844 0 0 Η 0 OH •0 0 _ -C-R1-C-NHC-C-〇-R4-〇- CC-NH-c-r1-c-nh-ch-r5-nh-L R3 R3 m CO-R2 q- ο Formula (IV) wherein η is in the range of from about 15 to about 150, and m is from about 0.1 to 0.9. Within the range; p is in the range of about 0.9 to 0.1; and wherein R1 is -CH2-N(CH2C02H)-R6-N(CH2C02H)-CH2-, wherein R6 is independently selected from (C2-C12) alkylene, ;?-C6H4, (C2-C4) alkoxy (c2-c4) alkylene, CH2CH2N(CH2C02H)CH2CH2 and the structure of formula (II) (wherein R7 is selected from hydrogen, (C^-Cu) alkyl, a group consisting of a protecting group) and a combination thereof; R7 R7 R7 —HC—N—CH— ^ —HC—N—CH—CH 2 or a H 2 C—HC—A—CH—CH 2 —H 2 ?h 2 ch 2 cooh cooh cooh

COOH COOH c〇〇H Formula (II) R2 is independently selected from the group consisting of hydrogen, (Cl_Cl2) alkyl or (C6_Cl()) aryl and a protecting group; V in each η unit is independently selected from hydrogen, (c丨_C6)alkyl, (c2_C6) dilute, (c2-c6)alkynyl, (C6_Cl0)aryl (Cl_C6)alkyl, _(CH2)2SCH3, ch2oh, ch(oh)ch3, (CH2)4NH3+, (ch2) a group of 3nhc(=nh2+)nh2, 4-indolyl imidazolinium, ch2COO_, (CH2)2CO〇- and combinations thereof; R4 is independently selected from (C2_C2()) alkyl, (C2_C20) Alkenyl, (c2-c6) alkoxy (c2_Ci2) alkyl, CH2CH(0H)CH2, CH2CH(CH2〇H) '1,4:3,6-di-dehydrated a group consisting of a bicyclic fragment of hexitol, a fragment of 1,4-anhydroerythritol, and a combination thereof; and 140309.doc -6-201039844 R5 is independently selected from the group consisting of (C2_C4) alkyl. In another embodiment, the present invention provides a method of preparing nanoparticle, and is hunted by at least one polymer having a chemical structure of the formula (1) or (4) in which: D is dissolved in an aqueous solution; The metal ions of the group consisting of Ca'Mg2+, Mn2+, Co2+, Fe2+, and Fe3+, 2+ • Zn, and Nl are contacted to form a nanoparticle containing a non-covalent complex of the polymer and the transition metal ion. 〇 In another κ~ example, the present invention provides a method of delivering a load molecule to a subject by administering to the individual a renter of the present invention. In another embodiment, the present invention provides a method for preparing nanoparticles by the following steps: a) contacting the following in an aqueous solution under polycondensation conditions: 1) Formula (1) or (IV) of the present invention Chelating polymer; 2) selecting Ca2+, Mg2+, Mn2+, Co2+, Fe2+ and Fe3+, Zn2+,

a metal ion of a group consisting of Ni2 and Gd3 +; and Q 3) an aprotic polar solvent; b) forming a nanoparticle containing a non-covalent complex of the polymer and the metal cation in the solution; and - c) Nanoparticles were obtained from the solution by size exclusion. [Embodiment] The present invention is based on the discovery that a biodegradable metal chelating polymer can be obtained by incorporating a polyaminocarboxylic acid into a poly(esteramine) PEA backbone. Metal chelating polymers integrate metal cations without binding to individual metal affinity ligands. 140309.doc 201039844 The biodegradable metal chelating polymer of the present invention is structurally related to the known poly(acetic acid) PEA, but in the present invention, the basic group of diacid used in the solution polycondensation of pEA is known. The fraction is replaced by an EDTA type polybasic acid (i.e., polyaminoacetic acid). The monomer for the synthesis of the polymer of the invention prepared from a polyamino acid of this type is an equivalent di-hepatic' which interacts with a diamine under solution condensation conditions to form a bis(α-amino fluorenyl)- The alcohol s is a monomer to form a stilbene bond. Thus, during the polymerization, two carboxylic acid groups of polyaminoacetic acid are used to form the polymer backbone along which the iminoacetate group is present. The remaining unbound carboxylic acid groups of the in-line residues of the polyaminoacetic acid in the polymer are freely chelated with the metal cations in solution. Thus in one embodiment, the present invention provides a composition comprising at least one polymer selected from the group consisting of: a polymer having the formula of formula (I), Ο Ό Ό-CC -NH- R3 R4 Ο Ο Η Ο

II. II I II C-R1-C-NH-C-C-0-R3 Formula (1) wherein η is in the range of from about 15 to about 15 0; R1 is independently derived from -CH2-N(CH2C02H)-R6-N (CH2C02H)-CH2-, wherein R6 is independently selected from (C2-C12)alkyl, Ρ-(:6Η4, (C2-C4)alkoxy (C2_<:4) alkyl and CH2CH2N(CH2C02H)CH2CH2 Or a group of formula (II) wherein R7 is selected from the group consisting of hydrogen, (C^-Cu)alkyl and protecting groups, and combinations thereof; and 140309.doc 201039844 R7

COOH COOH R7 R7 —HC—N—CH— A —HC—N—CH—CH2-ch2 ch2 ch2 c〇〇h

COOH COOH COOH Formula (II) R3 in each n unit is independently selected from hydrogen, (Ci_C6) alkyl, (CrC6), (C2-C6), (C6-C1()) aryl, -(CH2)2SCH3, CH2OH, CH(OH)CH3, (CH2)4NH3+, (CH2)3NHC(=NH2+)NH2, 4-methylene. a group consisting of iron, ch2CO〇-, (CH2)2COO_, and combinations thereof; R4 is independently selected from (C2-C6) alkoxy (C2-C12) alkylene, (c2-C2) extended alkyl , (C2-C20) an alkenyl group, CH2CH(OH)CH2, CH2CH(CH2〇H), a double ring fragment of 1,4:3,6-didehydrohexitol of the formula (III), iota, 4 - fragments of anhydrous erythritol and combinations thereof;

A pea polymer of the formula (III) or a formula of the formula (IV): o o c-r1-c-nh-ch-r5-nh I , C-O-R2 II o

ο Ο Η Ο OH -C-R1—c—NHC—C·. a R4-〇-6-έ-NH R3 R3 Formula (IV) wherein n is in the range of from about 15 to about 150, m is in the range of from about 0.1 to 0.9; p is in the range of from about 0.9 to about 0.11; And wherein R1 is -CH2-N(CH2C02H)-R6-N(CH2C02H)-CH2-, wherein R6 140309.doc -9- 201039844 is independently selected from (C2-C12)alkylene, P-C6H4, (c2- C4) alkoxy (c2_c4)alkyl, CH2CH2N(CH2C02H)CH2CH2 and a group of formula (II) wherein R7 is selected from hydrogen, (CrCu)alkyl, protecting group, and combinations thereof; R7 R7 R7 —HC—N—CH——HC—N—CH—CH—OR—H2C—Η〒一 A—CH—CH2-

CH2 CH2 CH2 COOH COOH COOH

COOH COOH COOH Formula (II) R2 is independently selected from the group consisting of hydrogen, (CVCu) alkyl or (C6-C10) aryl and protecting groups; R3 in individual η units is independently selected from hydrogen, (C-C6) Alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C10) aryl (Cl_C6) alkyl, -(CH2)2SCH3, CH2OH, CH(OH)CH3, (CH2)4NH3+ , (CH2)3NHC(=NH2+)NH2, 4-methylene misophthalene, cH2CO (y, (CH2)2COCT and combinations thereof; R independently selected from (C2-C20) stretching base, C2-C2〇) stretching base, (C2-C6) alkoxy group (C2-C12) stretching group, CH2CH(OH)CH2, CH2CH(CH2OH), formula (III) 1,4:3,6 a group consisting of a bicyclic fragment of dihydrohexitol, a fragment of i,4_anhydroerythritol, and combinations thereof; and R5 is independently selected from the group consisting of (Cj-C:4)alkyl. The chelating polymer is biodegradable and can be water soluble. The metal chelating polymer of the present invention can have a counterion (e.g., Na and κ counterion) associated therewith to form a salt. When the two amber 酴f type τη is formed, 140309.doc -10· 201039844 The I) or (IV) metal chelating polymer may contain a quinone imine unit as a cyclized dehydrated product of polylysine. The polymer of the present invention then comprises a chemical structure as shown in formula (V):

-NH*C—C-〇-R4-〇—c—C—N " r3 R3 I , C 一 H_ "C-CH2 R7 -N—CH— 0 :N—C—C—〇'R4 - 〇-R3 CC-NH R3 Ο ❹ As used herein, the term linear or branched hydrocarbon group of a bond. As used herein, "alkyne basic inventive metal chelating polymers may optionally be associated with a counterion selected from the group consisting of nano and unloaded. For example, the polymer may be associated with a nano ion to increase the polymer or contain the present. The invention is insoluble in the composition of the metal-integrated polymer. The polymer of the invention may be stored in the form of a free acid or a metal salt such as a metal salt. The protons in the pendant iminoacetic acid group may be via Na ions or K. The ionic moiety is partially or completely substituted to form a salt. As used herein, the term "aryl" refers to an ortho-fused bicyclic carbon represented herein as a phenyl group or having about 9 to 10 ring atoms, at least one of which is an aromatic ring. The structural formula of the ring group. In some implementations, one or more of the ring atoms may be substituted with one or the other of hydrazine, a gas group, a functional group, a trimethyl group or a trimethoxy group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and schlossylphenyl. As used herein, the term "alkenyl" refers to a structural formula herein meaning a branched hydrocarbon chain containing at least - an unsaturated bond in the main chain or in a side chain. "Non-cut" means "having one or more carbon-carbon doubles" means a straight or a bonded tobacco group having at least one carbon-carbon bond. As used herein, "an aromatic group of an aryl group" has a carbon atom in the range of 6 to 14

In the composition of the present invention, the metal chelating polymer of the household and the "A" is a polycondensate. In the description of the reduction and the object I, the four (4) and "P" in the formulas (IV and V) are defined as irrational numbers. In addition, since in any of the polycondensates ten "m" and p" will each have a range - the range of monomer residues that cannot be joined by a pair of integer bounds: All bis-amine groups The base diol - the known - one s day ω and the oriented amino acid (for example, lysine) monomer residue (ii) are both ώ $ π I μ Α ) ^ refined from the polyamino acid early body residues (iu) Connect to themselves or connect to each other. Therefore, only the centroid-out linear combination of seven i-in-u (or u-in-i) and η_(1)-u 1 in each of these combinations is based on the disulfide acid residue (4) Connect to yourself or connect to each other. Each polymer bond is thus a statistical (but non-random) bond from a single monomer and a monomer residue that is prepared. However, 'in general, for polymer chains having any actual average molecular weight (ie, sufficient average length), the ratios "m" and "ρ" of the monomer residues in formula (5) are not integers (reasonable Integer). Moreover, for all condensates of polydisperse copolymer chains, the number of all of the bond averages i, Η and Hi (i.e., normalized to average bond length) will not be an integer. The rate of migration is only an unreasonable value (that is, any real number that is not a rational number). The term irrational number as used herein is derived from a ratio that is not a Μ form, where η and j are integers. + As used herein, the term "amino acid" * "α-amino acid" means an amine group, a carboxyl group, and a pendant R group (such as the Han 3 group as defined herein). 140309.doc •12 - 201039844 compound. As used herein, the term "biological amino acid" means that the amino acid used in the synthesis is selected from the group consisting of phenylalanine, leucine, glycine, alanine, valine, isoleucine, methyl sulfide. Amine acid or a mixture thereof. As used herein, the term 'directed amino acid' means a chemical moiety derived from the polymer bond of a cardiac amino acid such that an R group (eg, R5 in formula (iv)) is inserted into the polymer backbone. Inside the chain. The metal chelating polymer of the present invention can be prepared as a solution polycondensation product of polyaminoacetic acid derived dianhydride and ◎ diamine (in detail bis(α-amino acid)-diol diacetate) in an aprotic solvent. . The lipid bond inherent in the bis(01-aminomercapto)diester monomer and its derived polymer can be hydrolyzed by a biological enzyme to form a non-toxic deuterated product. In an alternative, at least one of the amino acids used in the manufacture of the metal chelating polymers of the present invention is a biological heart amino acid. For example, when r3 is CHJh, the biological amino acid used in the synthesis is phenylalanine. In the alternative where R3 is CH2CH(CH3)2, the polymer contains the biological & 〇 amino acid, L-leucine. Other biological alpha-amino acids such as glycine (when h), alanine (when R3 is CH3), proline (or arginine) can also be used by altering R3 in the monomers described herein. When R3 is ch(ch3)2), isoleucine (when the ruler 3 is CH(CH3)CH2CH3), phenylalanine (when -R, CH2C6H5) or guanidine thiocyanate (when R3 is -(CH2) ) 2SCtMf) and its combination. In another alternative embodiment, all of the various alpha-amino acids contained in the polymer used to prepare the polymer delivery composition of (10) of the present invention are biological alpha-amino acids as described herein. In another embodiment, the I invention provides a method of delivering a 140309.doc 201039844 or multiple loading agents to a portion of an individual's body. In this embodiment, the method of the invention involves injecting into a living body portion of an individual's body a composition of the invention formulated as a dispersion of polymeric nanoparticles wherein the at least one loading molecule is maintained in a complex with a metal ion (tetra) Towel. The injected naphtha "slowly releases the mismatched load molecule. The dispersion of the nanoparticle of the present invention may be injected parenterally, such as subcutaneously, intramuscularly, or injected into an internal part of the body such as an organ. Biodegradable. Nanoparticles are used as carriers for at least one (eg, two) different loading molecules to allow them to enter the circulation t for systemic dry and timed release. The invention is sized from about 10 nm to about 500 nm The polymer particles will pass directly into the cycle to achieve these objectives. Depending on the choice of the polymeric base component, metal cations and especially the polyamino acid included in the compositions of the present invention, the compositions of the present invention may be used. The biodegradable polymer can be designed to adjust the rate of biodegradation of the polymer to cause continuous transport of the loaded molecule over a selected period of time. Suitable protecting groups for the MPEA metal chelating polymer include toluene-based salts (eg, Tos-OH) Or another protecting group known in the art. Suitable 1,4:3,6-dihydrohexitols of the general formula (III) include sugar alcohols (such as d-sorbitol, D-mannitol) Or L_Ai Dinoitol-derived two dehydrated hexitols. Shuangshuisorbitol is currently the preferred M:3,6-d-dehydrated hexose for use in the manufacture of the OEG-based polymer delivery compositions of the present invention. Alcohol bicyclic fragment. The α-amino acid is L- 'polymer contains α_. In an alternative, R3 is CHeh and the phenylalanine used in the synthesis. In the substitution of R3 for CH2_CH(CH3)2 140309.doc • 14 - 201039844 Amino acid, leucine. Other α-amino acids can also be used by changing R3, such as glycine (when R3 is hydrazine), alanine (when R3 is CH3), proline (when When R3 is CH(CH3)2), isoleucine (when r3 is ch(CH3)-CH2_CH3), phenylalanine (when R3 is CH2-C6H5), and lysine (when R3 is . -(CH2) ) 4-NH2) or methionine (when R3 is. • Double _ (L ~ leucine M, 6-hexanediol diester monomer (named Leu (6)) used in the manufacture of the chain The choice of the alpha-amino acid (by selection of r3) and the choice of diol and the choice of the intrachain polyacetic acid residue in the polymer of the invention contribute to the determination of the electronic properties of the metal chelating polymers of the invention. , named in this article The polymer of Leu(6)-EDTA consists of alternating hydrophobic segments (i.e., Leu(6)) and a strong electrical segment (i.e., intrachain EDTA). The resulting polymer is water soluble. A metal having a fraction of 1:1 (metal: in-chain EDTA) neutralizes the intra-chain EDTA group and thus the metallized polymer becomes a chain of alternating hydrophobic segments and neutral polar segments. The resulting metal is obtained using the method of the invention The polymer is readily condensed into particles (in this process, it captures Q with metal binding properties, any premixed load molecules). In addition to imparting biodegradability and biocompatibility, the bis (alpha, amino acid glycol diester moiety amino acid residues of the polymers of the invention can be selected to impart metal-bonded (or neutral) polarity The polymer has different biophysical and biochemical properties. For example, 'the Arg(6)- or Lys(6)EDTA is produced by substituting Arg or Lys for the Leu in the previous examples, and the polymer of the present invention is alternated. The positively charged section and the negatively charged section are formed and thus have a neutral charge and have a polarity. The polymer has a weak interaction with the self-denatured poly(nucleic acid). However, after metal chelation, the negatively charged chain The inner EDTA segment passes through 140309.doc •15-201039844 and 'generates a cationic polymer that interacts with the negatively charged poly(nucleic acid) via the positively charged Arg(6) segment (Cul〇mbiC inteFaetiQn And a strong interaction with the poly(nucleic acid) via a metal-mediated ionic bond between the EDTA segment and the poly(nucleic acid) in the metallization chain. Thus, in this example, Arg or Lys is substituted for the above Sufficient in the polymer of the invention (if Need) to impart greater stability in the load with negative electrode loading molecules. Instead, replace the above example Leu(6)_EDTA with Asp or Glu

The polymers of the invention are most suitable for loading cationic polar loading molecules. Replacing Leu in the above example Leu(6) EDTA with Ser, Thr, Asn, Gin, and combinations thereof, makes the metallized polymers of the present invention most suitable for loading neutral polar or poly(enzymatic) supported molecules, such as sugars and heavy Glycosylated protein. In addition to selecting intrachain alpha-amino acid residues to characterize specific loading molecules

The polymer (formula lb); and the solubility of different polymer chains. For example, a rigid diol (isosorbide, DAS) produces a water-insoluble and shorter aliphatic diol or a hydrophilic 1,4-anhydroerythritol-donating polymer (Formula Ic) that is hydrophilic and water soluble. Sex. a this can be made 丫舆Z cake. Xuan + 璺μ ΰΓ six 仏 ^. 丄 & ________

Non-limiting feta of (NTA), ethylenediaminetetraacetic acid (EDTA), and ethylenediamine-based polyamines: pentaacetic acid (DTPA), nitrogen triacetic acid > TA), ethylene glycol-double (2_Aminoethyl Ether)_ I40309.doc -16- 201039844 Stupid tetraacetic acid (EGTA), iminodiacetic acid (mA) and the like. The synthesis of the dianhydride residues of these polyamino acids is illustrated in the examples herein. Dihydroxy anhydrides of DTPA and EDTA are commercially available. Use of an aprotic polar solvent such as N,N-dimethylacetamide (DMAC), dimethyl sulfoxide (DMS) and methyl-2-pyrrolidone (NMP) from diterpene and monoamine The solution is polycondensed to form the metal chelating polymer of the present invention. Depending on the molecular structure and hydrophobicity of the diamine and dianhydride used during the polycondensation, the obtained poly-Q complex is soluble in an aqueous solution or is hydrophobic (and therefore insoluble). The polymers of the present invention form coordination complexes with various metal cations due to the imidoacetate groups along the polymer backbone. Suitable transition metal cations suitable for forming metal coordination complexes with the metal-binding polymers of the present invention to form the "metallized polymers" of the present invention include, but are not limited to, Ca, Mn, Ni, Co, Fe (2 + And 3+) and Zn of their cations/in non-radioactive and non-imaging metals, the most important based on biosafety is Zn, followed by Ni. Metal ions suitable for use in the preparation of radioactive or imaged metallated polymers include radioactive metal isotopes such as lanthanum, cerium and lanthanum. In one embodiment, the eye shovel is preferably used in diagnostic applications to image the transition metal cations associated with the polymers of the present invention to Gd(III) and to produce the metal chelating polymer of the present invention. For DTPA. • Since the free imidoacetic acid group is positioned along the flexible polymer chain used in the compositions and methods of the present invention, the metal ions can be aligned at an optimal position relative to the binding site on the surface of the loaded molecule. Thus, the loaded molecule can be non-covalently bound to the polymer via the resulting metal affinity complex. In other embodiments, the free -NH2 end of the polymer molecule can be deuterated to ensure negative 140309.doc 17 201039844 and the carrier that is not attached to the polymer only attaches to the free end via a metal affinity complex. A transition metal cation in which the imidoacetic acid group is bonded to the initial phase produces a composition referred to herein as a "metallized polymer" in which at least one free valence of the chelate metal is available for bonding to the metal cation. Affinity therapeutic load molecule. As described in more detail below, the amino acid in the polymer backbone allows for the stabilization of the power of the gold polymerized composition and the load molecules in the nanoparticles of the compositions. ~ Suitable loading molecules which can be misaligned by the metallated polymers of the present invention include polar bioactive agents such as pharmaceuticals; "biological agents" and His-tagged molecules. The term "biological agent" as used herein encompasses both natural and synthetically produced proteins, peptides, polyamino acids, fusion proteins and polynucleic acids, including vaccine antigens, such as those described herein as SEQ ID NOs: 1-8. The term "macromolecular biologic" as used herein includes biological agents that have a unique three-dimensional folded structure of biologically active opsin molecules (e.g., proteins, polypeptides, and polynucleic acids). It has also been found that the biological activity of vaccine antigens also depends on the preservation of the natural three-dimensional folded structure of the molecules present in the maternal pathogen in the vaccine formulation. As described more fully below, it has been discovered that the electrical power of the metallated polymers of the present invention can capture and stabilize biologics and macromolecular biologics as well as lipophilic loading molecules containing negative polar domains from aqueous solutions. The presence of at least one histidine residue in a bioburden molecule (e.g., a His-tagged protein, peptide antigen, or fusion & construct) is an important factor in facilitating binding of the loaded biologic formulation to the polymer. The amine group of the loaded biologic or I40309.doc -18- 201039844 The carboxy terminal His (i.e., His tag) results in a specific modification of the binding of the loaded molecule to the metal ion in the metal affinity complex. Thus, in one embodiment, at least 1 to about 10 contiguous His residues are incorporated in one or both of the amine and carboxy termini (eg, about 6 His residues (ie, "six His" Label, label") as a label to ensure binding efficiency. If a His label is added, the final label (for example, nano particles) is allowed to retain His label and metal chelate (for example, Ni or Zn metal chelate) Q Because the pK value of the histidine group that is bound to bind is in the neutral range, it is expected that the binding of the loaded biomolecule to the polymer may occur at a pH of about 7. However, the actuality of the individual amino acids The pK value can vary greatly depending on the influence of the adjacent amino acid residues. Various experiments have shown that depending on the protein structure, the pK value of the amino acid can deviate from the theoretical pK value by up to a unit of pH. Therefore, the pH is about A modified combination of 8 is usually achieved. The addition of other metals present in the biomolecule to the amino acid (such as cysteine and tryptophan) also contributes to the metal bond. In addition, it is an established base metal bond. Biological category of protein The agents are not necessarily suitable for use as bioburden molecules in the compositions and methods of the present invention. Crystallizers typically use transition metal-bound protein analogs as the basis for structural resolution in structural studies. This procedure is referred to as "homotype replacement." And the discovery that regardless of whether the metal binding site in the molecule has biological function, all proteins and polynucleic acids are at least weakly bound to the transition metal (M Babor et al., (2008) 70:208-217 and the World Wide Web interscience. Wiiey c 〇m/jpages/〇887_ 3 5 85/8叩?11^/On the supplements and 1^¥&115, etc., price/〇7 CTzew (2005) 10:476-482). 140309 .doc -19· 201039844 In the present invention, it has been found that all biological agents (including macromolecular biological agents) have a weak affinity for the transition metal and the main chain amino acid of the composition of the present invention to capture and retain the metallized polymer of the present invention. And the supported molecules in the nanoparticles prepared by the polycondensation process of the present invention. The metallized polymers of the present invention provide an affinity for stabilizing the supported particles. Surprisingly, it has been found that even in the form of macromolecules The lipophilic bioactive molecules may also be chelated by the metallated polymers of the invention. The bioactive molecules are characterized by having a cLogP in the range of about 2 to 6.0 and characterized by the presence of an unsaturated region (including aromatics). The group) and 2) the negative electrode composed of the ruthenium- and S- and N-groups. The metallized polymer of the present invention which has been mismatched with the lipophilic load molecules can also be formulated into nanoparticles using the process of the invention for polycondensing nanoparticles. Examples of such macromolecular lipophilic pharmaceutical compounds which are preferred for the misalignment of the metallated polymers of the present invention include, but are not limited to, Taxane, such as paclitaxel and docetaxel. And the muscarinic compound (Hmus c〇mp〇und) 'such as sirolimus, Everum Hmus and Biolimus. More specifically, paclitaxel has a cLogP of about 3.5, and thus has a macroscopic property of a highly lipophilic drug having extremely low water solubility. However, examination of its surface at the atomic level shows that although the molecule is hydrophobic at the macromolecular level, there are polar domains provided by aromatic groups and oxygen atoms. These polar domains as seen on the surface of the hydrophobic molecule are responsible for the binding of paclitaxel to the cavities in its dry protein (p-tubulin), which is lined with polar and hydrophobic amino acid side chains. The affinity of these compounds to weakly bind to the free coordination sites in the metallated poly(140309.doc-20-201039844) of the present invention (i.e., the sum of the microaffinities) results in the nanoparticle of the metallized polymer of the present invention. The particles (4) are lipophilic loaded with molecular stability. As another example, 'Rapamycin (sirolimus) is among the most hydrophobic drugs currently used, which has a cLogP of about 5'5 and _ hydrophobicity is more hydrophobic than paclitaxel. About 10 times higher. However, lysin carries several microdomains with unsaturated bonds (similar to the aromatic region on paclitaxel) or a pair of isolated buns around the oxygen atom ( (e.g., in cedarol). It is believed that these microelectron regions are important at the molecular level for the biospecificity of the directed rapamycin affinity for its protein. Because it represents a concentrated source of strong multivalent ionic bonds, metal ions are ideally suited for search and lock-in even in the most hydrophobic clinically applicable compounds (eg, ligands that specifically bind to larger dry proteins in vivo) , the micro-polar region also visible on the compound). Another example of a suitable loading molecule suitable for loading in the metallated polymer of the present invention is a clear albumin (SA), which is well known and well known in the art. Sa ” has the following chemical and biological properties, making it particularly suitable for inclusion in metal-integrated polymer coatings, implanted characters or particles (as shown in Example 5 herein): a 'high affinity metal binding site' 2 Angiogenic blood vessels around the tumor

'Companion to dryness characteristics' and 3) Southern blood compatibility (causing the use of particles loaded with SA for intravenous delivery). When f is used as a loading molecule in the composition of the invention, SA may have several therapeutic uses due to the high blood phase of the valley: 丄) used as an antidote to metals, 2) used as lipophilic (thus penetrating cells) Toxins (eg, plant anti-purple molecules (8) _ 140309.doc 201039844 defense m〇iecule), such as antidote '3 of ^..?) used as a blood-borne transport agent for natural hydrophobic molecules (fatty acids, steroids 1 _ ), or 4) Used as a medicament for maintaining blood osmotic pressure (important for the exchange of blood volume with other body fluids), which is suitable for the metallization of the bioactive agent of the present invention. Including, but not limited to, a 八; music, therapeutic biological agents, such as 姨 素 A A growth hormone and blood drop; treatment and dry antibody and pot active fragments; known therapeutic blood factor +, Such as coagulation factor 丨 and protein and cool protein antigens such as those suitable for inclusion in subunit vaccines. Further, peptides (including those containing a pathogenic epitope of a secondary unit vaccine) can be incorporated into the metallized polymer compositions of the present invention. In particular, the amino acid sequence comprising a pathogenic epitope can be incorporated into a metallized polymer composition of the invention in a subunit vaccine formulation wherein the unique three-dimensional folded structure of the epitope is preserved. Non-limiting examples of such antigenic amino acid sequences include the ones described herein as SEQ ID NOs: 1-8 in Figures 5-12. Formulations of supported metallated polymers are diverse and include implants, coatings, and nanoparticles, such as vaccine formulations. For example, in one embodiment, the present invention provides a method of formulating a metallized polymer of the present invention into nanoparticle using a solution polycondensation technique that avoids the need for emulsion techniques typically used to formulate polymer particles. As described in Examples 4 and 5 herein, as the final step of polycondensing the metallized polymer, the metallized polymer of the present invention can be readily & formulated into nanoparticles whether or not otherwise mismatched with one or more load molecules. In addition, the polycondensation process of the present invention produces particles which are more dispersible in aqueous environments than particles based on the more hydrophobic first generation PEA. 140309.doc • 22- 201039844 sub-the diol used in the manufacture is, for example, Chucc, Aliphatic dicarboxylic acid as disclosed in Katsarava R US 6,503,538 B1. The method of the present invention for preparing a nanoparticle of a metallized polymer loaded with a support comprises the steps of: a) preparing a homogeneous mixture of a supported molecule and an aqueous solution of the polymer of the invention; b) by passing to a supported molecule  : the rapid addition of aqueous metal salt in the liquid to prepare the loading molecule, the transition metal salt solution; and em by adding a solution of a) to the Ο at room temperature under _

The nanoparticles are produced in b). (Recovering, for example, from such a technique as described in the art and recovering the nanoparticles from the reaction solution by size exclusion filtration, dialysis or centrifugation and washing techniques as described herein). The metallized polymer of the present invention having integrated load molecules is applied as a viscous liquid coating to various types of particles using a variety of techniques known in the art, such as spraying, dipping, and the like. Externally. For use as a coating, the load molecules included in the present invention are selected from, but not limited to, blood factors, including serum albumin, transferrin, antibodies, and active fragments thereof; and such load molecules a singularly labeled fusion construct. As is known in the art, the coatings can also be applied to at least a portion of the exterior of various types of solid articles used in the treatment of medicinal materials for enhancing the coating of the edge coating particles. Or money or histocompatibility of a medical device. μ In another embodiment, the invention may be administered to a subject for the purpose of metal detoxification and/or wound care. A sounding substance, which is formulated to be administered alone or in the form of a plant or a particle: an adjuvant for the treatment of a bioactive agent. ^ 140309.doc -23- 201039844 In another example, the metallized polymer of the present invention may be used. Formulated as coating implants and particles to be used for presenting and/or delivering therapeutic drugs and biological agents. s 'The metallized polymers of the invention can be combined with drugs and biological ligands (such as antibodies or stem cells) A surface marker, a specific receptor or other ligand of a protein docking site) is co-loaded, wherein the biological coordination system is used to deliver the composition and the chelated drug to a target cell or cell type (such as a cancer cell type) The drug may be selected to kill the natural coordinating molecule, block the anchorage of the natural coordinating molecule, or prevent replication of the molecule in the target tissue or cancer cell. In another embodiment of the invention, the polymer of the invention The particles are co-supported with a supported paramagnetic or ferromagnetic metal and a biological ligand as described herein. A paramagnetic or ferromagnetic metal system is used to deliver a composition of a composition to a biological ligand after parenteral injection. Device Diagnostic imaging of tissue, tissue or cells. Methods of using such diagnostic compositions are well known in the art. In another embodiment, a radioactive metal as described herein is chelated by a metal chelate polymer of the invention and a second is used. Molecules (targeting ligands known in the art and described herein) are used for tissue or cell targeting. For example, a radioactive metal can target stem cells in a malignant tumor by incorporation of a ligand ( An antibody, such as an antibody that specifically binds to a cell surface marker thereon, such as an antibody that specifically binds to CD20, kills stem cells. In another embodiment of the invention, nanoparticles for diagnostic imaging are as described herein. Diagnosing metal ions (eg, Gd3+) and ligands that specifically bind to target cells, organs, or tissues are co-loaded. Methods for performing Gd imaging are well known in the art and include, but are not limited to, in vivo magnetic resonance imaging 140309 .doc -24-201039844 (MRI), wherein the diagnostic composition is parenterally injected for diagnostic imaging and the dry coordination system known in the art and described herein is used for tissue or Stem cells to. Thus, in one embodiment, the metal chelate polymer of the present invention is chelated with a diagnostic genus to form a diagnostic composition for in vivo administration of a desired target cell, organ or group. The polymer composition can be readily biodegraded and discharged. Thus, the inventive diagnostic compositions prepared using the metal meal polymers of the present invention avoid long-term tissue accumulation of chelating toxic ions and can be formulated into nanoparticles using the polycondensation methods described herein. In another embodiment, the polymer of the invention is conjugated to a bioactive agent via a polymer end group and/or conjugated using a terminal group to obtain an ABA type block system, "中中 B is a formula (1) or formula (iv) The polymer and a segment are selected from compounds such as peg (polyethylene glycol or polyethylene glycol), polysaccharides, lipids, biomacromolecules (such as polypeptides or poly(nucleic acids)) and active agents. In this case, the 6-block polymer macromolecular chain preferably has an equal or equal number of active end groups (amine oxime or anhydride) (other conjugate sites are carboxy groups attached along the side of the macromolecular chain). The balancing technique achieves a B block synthesis for incorporation into a hydrazine block chelating polymer having an equal or equal number of identical end groups, wherein 'a pre-calculated excess is introduced at the beginning of the polymerization as described herein. A difunctional monomer used in the polycondensation of a chelate-polymer (i.e., a diamine or an activated polyacid) is invented. When an excess anhydride end group is used, the method becomes complicated because according to the Maldi-TOF spectrum. Monitoring produces a large number of polymeric rings (macrocycles). However, 'discovered The addition of an inorganic base (for example, K2C03) significantly reduces the reaction rate and allows better control of the Mw of the resulting linear oxime block polymer. 140309.doc •25· 201039844 The integrated polymer molecule can have end-group conjugates ( Optionally, via a linker, a bioactive agent attached thereto. For example, in one embodiment the 'chelating polymer is included in the polymer-bioactive agent end group co-roller of formula VIII:

9 〇Η 0 OH loo C~R1-C-NH-C-C-〇-R4-〇-CC-NH-hC-R1C-R8-R9 R3 R3 Formula (VIII) where n, Rl, Han 3 and R4 As described above, R8 is selected from the group consisting of -o-, _S-, and NR1G where rH is H or (Ci_C8)alkyl; and R9 is a biologically active agent as described herein. To obtain a vaccine formulation, in one embodiment, an amino acid sequence comprising at least one pathogenic epitope that maintains its natural conformation is unbound by the intrachain residues of the polyaminoacetic acid in the polymer of the invention. A carboxylic acid group (i.e., R3 in the chelating polymer or metallated polymer of the present invention) is attached to the chelating polymer of the present invention. Alternatively, in a vaccine formulation, the unbound carboxylic acid groups of the intrachain residues of the polyaminoacetic acid in the polymer of the present invention are freely chelated with the metal cations in solution to form a metallated polymer. The metal cation promotes further attachment of the metal-bound amino acid in the pathogenic cardinogen. The emulsion technology that is typically used to form polymer particles can readily obtain nanoparticle from a metallized polymer vaccine formulation directly from a solution containing the polymer. A method of formulating a vaccine as a nanoparticle using the chelate (e.g., metallized) polymer of the present invention is described in Examples 8 and 9 herein. In another embodiment, which is described in detail below, R9 of the terminal conjugated formula is a biologically active agent, such as one or more of various immunostimulating adjuvants. 140309.doc •26·201039844. The immunostimulatory adjuvant includes a drug such as 17 m Imiquimod; a lipid such as QS-21; a nucleic acid such as a dsRNA analog poly I: poly C; or an immunostimulatory protein such as GM-CSF. The particular desired immunostimulatory adjuvant suitable for conjugation to the polymeric end groups of the present invention enhances the utility of the chelating polymers of the present invention formulated as vaccine compositions which are arranged according to the type in Table 6 below. Table 6 Adjuvant name _ type ^ Calcitrol drug imiquimod drug Loxoribine drug poly rA: poly rU drug S-28463 SM360320 drug Adjumer polymer CRL1005 polymerization PLGA, PGA and PLA Polymer Pluronic L 121 Polymer Ο PMMA Polymer PODDS Polymer SAF-1 Polymer SPT Polymer Avridine Lipid. Bay R1005 Lipid DDA Lipid DHEA Lipid DMPC Lipid DMPG Lipid D-Murapalmitine Lipid 140309.doc -27- 201039844 DOC/Aluminum Complex Lipid ISCOM Lipid Iscoprep 7.0.3 Lipid Liposome Lipid MF59 Lipid Montanide ISA 51 Lipid Montanade ISA 720 Lipid Mulapamistatin Lipid Nonionic Surfactant Liposome Lipid Polysorbate 80 Lipid Protein Spirochetes Lipids 85 (Span85) Lipid Stearylsyl Tyrosine Lipids Theramide Lipid Gerbu Adjuvant Lipid/Sugar QS-21 Lipid/Sugar QuilA Lipid/Walter Reed Liposomal Lipid/Algal Glucan Sugar Algammulin Sugar gamma Inulin Glycerin GMDP Sugar ImmTher Murametide Sugar Pleuran Sugar sulphate-MDP Sugar Adju-Phos Salt Alhydrogel Salt Calcium Phosphate Gel Salt Adsorption Hydroxide Gel HPA (Rehydragel ΗΡΑ) Salt-adsorbed aluminum hydroxide gel LV salt cytokine-containing liposome biological preparation 140309.doc -28- 201039844 GM-CSF biological preparation immunoliposome (DRV) containing anti-co-stimulatory molecule antibody biological agent single-stranded DNA Biologics Single-stranded RNA Biologics Double-stranded DNA Biologics Double-stranded RNA Biologics Interferon-γ Biologics Interleukin-12 Biologics Interleukin-1Β Biologics Interleukin-2 Biologics Interleukin-7 Bio Formulation LT-OA (LT-oral adjuvant) Biological preparation Sclaro-like biological preparation Sendai proteoliposome, lipid matrix preparation containing Sendai, Ty particle biological preparation, squalene burning oil, Example 1 in this example, which is described in the vaccine formulation An example of a method for immunostimulating adjuvant end groups in the preparation of nanoparticles. Alternatively, as shown in the following structural formula (IX), a linker-Χ-Υ- may be inserted between R8 and the bioactive agent R9 in the molecules of the structural formulas (1) and (IV), wherein X is selected from the group consisting of the following groups Group consisting of: (Ci-Cu) alkylene, (C2-C8) alkoxy (C2-C2Q) alkyl, substituted alkyl, (C3-C8) cycloalkyl, substituted ring Alkyl, 5-6 membered heterocyclic ring system containing 1-3 heteroatoms selected from the group consisting of hydrazine, N and S, substituted heterocyclic ring, (C2-C18) alkenyl group, substituted alkenyl group, alkyne a substituted alkynyl group, a C6 and C! aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, an alkylaryl group, a substituted alkylaryl group, an arylalkynyl group a substituted arylalkynyl group, an arylalkenyl group, a substituted aryl fluorenyl group, an arylalkynyl group, a substituted arylalkynyl group, and its _ taken 140309.doc • 29- 201039844 From the group Η, F, Cl, Br, I, (CVC6) alkyl '-CN, -N02 '-OH, -CKq-CO alkyl, -SCCVCO alkyl, -SK^OKCk c6) alkyl, -SKC^CCVCe)alkyl], -CthOKCVCO alkyl], CF3, -OKCOHCVCO alkyl], -S(02)[N(RnR12)], -NH[(C=0)( C!-C6)^ > -NH(C=0)N(RnR12) '-N(RnR12); wherein Rn and R12 are independently H or (Ci-CJ alkyl; and Y is selected from -O-, -S-, -SS-, -S(O)-, -S(O2)-, -NR10-, -C(=O)-, -OC(=O)-, -C(=〇)0- , -〇c(=〇)NH-, -NR10C(=O)-, -C(=0)NR10-, _NRi〇C( = 0)NR10_, _NR10C(=O)NR10- and -NR10C(=S ) NR10- group of groups. ο ο —C-R1 C-R8-XY-R9 J n

9 9 Η Ο ο Η C-R1~c-nh-cco-r4-occ-nh R3 R3 Formula (IX) In another embodiment, the chelating polymer of the present invention can be used in the design of an ABA type block system. 'wherein B is a polymer of formula (1) or formula (IV) and the A block is selected from, for example, PEG (oligoethylene glycol or polyethylene glycol), polysaccharides, lipids, bio-knives (such as polypeptides or Poly(nucleic acid)) and a compound of a bioactive agent. The rhodium block polymer of the present invention was formed by the terminal conjugate technique as described in Example i 0 herein. In the process for preparing the oxime block acceptor of the present invention using the chelating or metallated polymer of the present invention, and in all of the terminal conjugated eclipses using the polymers, the ruthenium polymer macromolecular bonds are preferably equal. Reactive end group amine or anhydride (other co-sites are carboxyl groups along the side of the macromolecular chain). In an end group conjugate (whether in the simple 140309.doc • 30· 201039844 end group conjugate as described above or in the formation of the oxime block polymer of the present invention) Synthesis of a number of active end groups of the chelating polymers of the invention. For both of these procedures, an unbalanced technique is employed in which a pre-calculated excess of a difunctional monomer used in the polycondensation of the chelate polymerization of the present invention as described herein is introduced at the beginning of the polymerization (ie, two Amine or activated polyacid). This method becomes complicated when an excess of anhydride end groups is used because a large amount of polymerized rings (macrocycles) are produced according to the Maldi-TOF spectrum. However, it has been found that the introduction of an inorganic base (e.g., K2C03) significantly reduces the rate of reaction and allows for better control of the Mw of the resulting linear bismuth block polymer. In one embodiment, PEG is introduced as the A block in the bismuth block polymer to increase the solubility of the highly insoluble loaded drug which is retained in the coordination complex by the metallated polymer. The metallized polymer with the insoluble loading drug forms a B block flanked on both sides as a PEG molecule with increased solubility as the A block. It has been surprisingly found that in this embodiment of the invention, the ρ size of the nanoparticles formed from the ruthenium block polymer is substantially reduced compared to the size of the nanoparticles prepared using other embodiments of the invention. . For example, nanoparticles of the rhodium block polymer having a range of from about 50 nm to about 100 nm (e.g., about 68 nm) have been obtained. • The invention is further illustrated by the following non-limiting examples. Example 1 Material reagents were all derived from Sigma-Aldrich diethyltriamine pentaacetic acid dianhydride (DTPA-DA, 98%), ethylenediaminetetraacetic acid dianhydride (EDTA-DA, 98%), ethylene glycol-double (2-Aminoethyl ether tetraacetic acid (EGTA, IDRANALTM IV) is used as it is. For example, Geigy, JRA-140309.doc • 31 - 201039844 G. in Fr. Patent 1,548,888 (Cl.C07d); C/zew (1969) 71:81380q reported that other dianhydrides (such as EGTA dianhydride) can be prepared by dehydrating the parent tetrabasic acetic anhydride in pyridine. Iminodisuccinic acid (IDS) disodium salt ( Baypure CX100 G, 77%) is a free sample from Obermeier GmbH & Co, Bad Berleburg, Germany. Amino acids (L-leucine, L-phenylalanine, glycine, L-arginine, L- The lysine) and the diol (1,3-propanediol and 1,6-hexanediol) were obtained from Sigma-Aldrich. Anhydrous solvent dimethylamine (EMD Chemicals, Inc, NJ), hydrazine, hydrazine- Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hydrazine, hydrazine-dimethylacetamide (DMAc) (Fisher Scientific) and other solvents C, 2-propanol, methanol, toluene ( Spectrum Chemicals, CA) From commercial sources. Material characterization The chemical structure of monomers and polymers was characterized by standard chemical methods. NMR was recorded by a Bruker AMX-500 mass spectrometer (Numega R. Labs Inc. San Diego, CA) operating at 500 MHz. 1 NMR spectroscopy in the spectrum. Solvent CDC13 or DMSO-i/6 (Cambridge Isotope Laboratories, Inc., Andover, MA) was used as an internal standard with tetramethyl sulphur (TMS). In automatic Mettler-Toledo The melting point of the synthesized monomer was measured on a FP62 melting point apparatus (Columbus, OH). The thermal properties of the synthesized monomers and polymers were characterized on a differential scanning calorimeter (DSC) (Mettler-Toledo DSC 822e). The sample was placed in a dial and measured under a nitrogen flow at a scan rate of 1 〇 ° C / min. Equipped with a high pressure liquid chromatography pump, Waters 2414 refractive index detector 140309.doc •32· 201039844 ( Refractory index detector) 515 gel permeation chromatography (waters

Associates Inc. Milford, ΜΑ) measures the average molecular weight and weight average molecular weight (Mw and Μη) and molecular weight distribution (Mw/Mn) of the synthesized polymer. The eluent used was a 0.1% LiCl solution (1.0 mL/min) in hydrazine, hydrazine-dimethylacetamide (DMAc). Two Styragel® HR 5E DMF columns (Waters) were attached and calibrated with polystyrene standards. Monomer Synthesis: The synthesis of the biodegradable polyaminocarboxylic acid-containing polymer of the present invention involves two basic steps: 1) a bis-nucleophile (bis(α-aminoindenyl)-monolol-- (Synthesis of a di-p-benzoic acid salt of a compound of the formula VI); and 2) a polycondensation of a solution of the monomer obtained in the step 1) with a tetracarboxylic dianhydride. Η Ο 9 Η

Tos〇-.H3N+-CC-〇-R4-〇-CC-NH3+.Tos〇·R3 R3 Formula (VI) Synthesis of acid salt of bis(α-amine-based warm) diester (Formula VI) Procedure for the preparation of a diester of the formula (VI) by a procedure of the disclosed procedure: α-amino acid (0.1 mol), p-toluenesulfonic acid monohydrate (ou ^οΙ) and diol (0.05 mol) in 150 mL of benzene The suspension was stirred and refluxed in a Dean-S. Tark condenser until 3.6 mL (0.2 m) water (12-24 hours) was precipitated. The heterogeneous reaction mixture was cooled to room temperature and the solid product was filtered, washed with toluene and dried under reduced pressure. The monomers synthesized in the form of di-p-toluenesulfonate using this procedure are designated herein as follows: Bis-(L-leucine-branched)-1,6-hexanediol diester ([_]^ιι(6) )-2Τ〇8〇ίί;), bis-(L-stactylamino)^4:3,6-bis-desorbed sorbitan ester (L_140309.doc •33- 201039844

Phe(DAS)-2TosOH) bis-(glycine)-l,4-anhydroerythritol diester, (Gly(THF)-2TosOH).

The yield and melting point (Mp) are consistent with the published data. (Katsarava et al., 丄Po/jvw. 5W. Par, 儿·尸〇/少w. C/zew. (1999) 37:391-407 ; Z. Gomurashvili et al., J. Macrowo/. Sci. -Pure .Appl. Chem. (2000) A37:215-227; ZD Gomurashvili et al., US 20070282011). Synthesis of bis(L-spermine fluorenyl)-1,6-hexanediester tetramethanesulfonyl sulfonate (Arg(6)-4TosOH) of formula (VII) Η Ο Ο Η ΤοβΟ'.ΗβΝ*—CC- 〇-(CH2)6~〇-CC-NH3+.TosO' (CH2)3 (CH2)3

NH NH C=NH2+.T〇sO' c=nh2+.t〇s〇- nh2 nh2 Formula (VII) A monomer having the formula (VII) is synthesized following the same procedure as described above, but using 0.22 mol of monohydrate p-Toluenesulfonic acid. For monomer purification, 5 g of crude monomer was dissolved in 30 mL of heated 2-propanol and filtered through filter paper to remove excess arginine. After storage in the freezer, the layer of viscous monomer is separated. This procedure was repeated twice and the final product was dried under vacuum overnight. The product was then redissolved in 1 g/mL water and lyophilized. A 75.9% yield of absorbing white material was obtained, melting point = 264-268 ° C (DSC, 5 ° C / min). Elemental analysis: C46H7〇N8016S4 (1119.35). Calculated value: C 49.36, Η 6·30, Ν 10.01. Experimental values: C 49.72, Η 6.53, Ν 9.96. 140309.doc -34- 201039844 Synthesis of bis-L-leucine _peG2〇0-diester bis-p-toluenesulfonate, formula (VI), wherein R3=CH2_CH(CH3)2, r4=peg 0 . Tos〇-.+H3N-CH-C-(〇-C ch2 \ CH(CH3)2 \ 9 H2-CH2j-〇-c-CH-NH3+.Tos〇- ?H2 CH(CH3)2 will be L-white Amino acid (17.46 g) (〇.133 mol), 26·53 g (〇i4 mol) monohydrate p-toluic acid and 11.25 mL (63.4 mmol) PEG-200 (Aldrich) ❹

Suspended in 19G mL of anhydrous toluene and mixed with overhead scramble (4). The solution was heated to reflux for about 8 hours and the precipitated water (48 mL) was collected in a Dean-Stek condenser. After standing at room temperature, a light brown-yellow oily layer was isolated. The solvent was then decanted, and the product was dissolved in 5 mL of 2-propanol and precipitated as an oil in 50 mL of hexane. The yield of the light brown orange oily crude product collected was 42 g. The 1G (iv) material was redissolved in 150 mL of hot benzene and then allowed to pass at 4. The armpits escaped as an oil overnight. The solvent was decanted and the product was dried in a vacuum oven at 6 Torr for "hours. L-Acetyl-PEG 2 〇 0-diester bis-p-toluene sulfonate: 5 〇〇 MHz brilliant R (DMSO- 4 ppm, δ): 0.89 [d,12H,CH_(C horse)2],i 6〇[m,4H,-CH_C/i>CH-], 1.74 [m,2H, -C7f-(CH3)2 ], 2.29 [s 6H, -Ph-Cif3], 3.50 [s, 4H, -OCO-CH2-CH2-〇-], 3.53-3.64 [m,m~10H, -0-CH2-CH2-0-] 4.〇〇[s, 2H, +H3N-C^-], 4.23. 4.34 [m,m, 4H, -OCO-CH2-], 7.14 -7.49 [d5d, 8H, Ph]5 8.33 [s, 6H , +H3N-] 〇 poly. Synthesis 1. Reaction study of polycondensation reaction 140309.doc •35- 201039844 Study on the polycondensation of EDTA-DA with the diamine monomer L-Leu(6).2TosOH Optimize the reaction parameters and increase the Mw of the product. 1. Effect of the base Triethylamine (TEA) is used as the base/catalyst. EDTA-DA is combined with 2 molar equivalent base (for L-leu(6) The reaction of each of the tosylate salts is 1 equivalent) and 4 moles equivalent (1 equivalent for each benzenesulfonate and 1 equivalent for each of the resulting free carboxyl groups formed for EDTA) The reaction was compared. Seen in Table 1 below. The results show that when the carboxylic acid of EDTA is occupied, the Mw size of the polymer is increased by more than two times by using a double increase in molar equivalent. Table 1 Effect of the amount of alkali on the molecular weight (Mw) of the base Alkali equivalent / Dihydride Mw MP PDI TEA 2 33028 29484 1.44 2.2 25263 23019 1.49 4 71322 83906 1.97 4.04 80944 89376 1.84 Solvent: DMF; reaction time: 24 hours; temperature: 20 ° C; [anhydride] = [diamine] = 0.9 Μ 1.2 The effect of temperature on the Mw of the polymer During the initial PEA EDTA-Leu(6) reaction at 60 °C, it should be noted that the color of the reaction mixture is significantly deeper, from pale yellow to dark amber, and the viscosity is low. In order to compare the effect of temperature on color change and attempt to achieve higher Mw, the reactions were carried out at 60 ° C, 40 ° C, 20 ° C and 〇 ° C. The results are listed in Table 2 below. Decrease, the color change of the reaction mixture is less significant and 140309.doc -36- 201039844 obtains a higher Mw product. This result indicates that the anhydride easily reacts with the diamine comonomer even at a low temperature, and an unforeseen side reaction which causes termination or inhibition of chain elongation occurs at a higher temperature. Table 2 Effect of reaction temperature on Mw Temperature (°C) Mw MP PDI 60 25,263 23,019 1.49 40 33,291 31,653 1.52 20 71,322 83,906 1.97 0 143,886 104,004 2.12 Solvent: DMF; reaction time: 24 hours; [anhydride] = [diamine] = 0.9 Μ 3.13. Kinetics As seen in the polycondensation experiments described above, the polymer achieved a maximum molecular weight in the first hour. The optimum temperature for the EDTA-diamine condensation reaction (see Table 3 below) is considered to be in the range of 0 °C to -20 °C. Table 3 Effect of reaction time on Mw

Reaction temperature (°C) Reaction time (hours) Mw MP PDI i'-'·"zi ·6·8·8·6 il 11 ΙΑ 1 85362 91053 3 77468 86122 6 80944 89376 8 73501 78543 1 65235 80843 1.85 3 71875 83906 1.97 5 70312 85485 1.9 8 72990 88742 1.94 24 71322 83906 1.97 20 40 2 20 32685 33291 30023 31653 1.52 1.52 24 25263 23019 1.49 Solvent: DMF; reaction time: 24 hours; [anhydride] = [diamine] = 0.9 Μ 3.1.4. Solvent Selection 140309.doc -37- 201039844 Compare the applicability of the aprotic polar solvents DMSO, DMF and DMAc in the polycondensation reaction. DMSO is the first solvent choice because EDTA-dianhydride is readily soluble in it. However, as the polycondensation reaction proceeds, the formed polymer is suspended in any of the three reaction solvents. The resulting Mw of the polymer obtained in each of the three reaction solvents is shown in Table 3. Both DMSO and DMF discolor the polymer and the use of DMSO causes a unique sulfurous odor. No disadvantages were observed when the reaction was carried out in DMAc: the polymer and suspension were off-white and odor free. As can be seen from the data in Table 4, the Mw of the polymer formed in DMF and DMAc is comparable, but DMSO is used to produce a polymer having a significantly lower Mw. Table 4 Effect of solvent on Mw Solvent Mw MP PDI DMSO 84722 84672 1.75 DMF 143886 104004 2.12 DMAc 122825 95264 2.03 Temperature: 0° (:; reaction time: 8 hours; [anhydride] = [diamine] = 0.9] ^ PEA EDTA Synthesis of -Leu(6) polymer (Formula U) · COOH,

' I Ο CH2 ο Η ο Ο Η 4c-CH2-N-(CH2)2-N-CH2-C-NH-CC-〇-(CH2)6-〇-CC-NH—I" , ch2 卜CH2 Jn COOH CH(CH3)2 ch(ch3)2 Formula la For the polycondensation, bis-(L-leucine fluorenyl)-1,6-hexanediol diester bismuthene phthalate (8.32 mmol, 5.734 g) was mixed with EDTA-DA (8.32 mmol, 140309.doc -38·201039844 2.133 g), followed by the addition of 4.69 mL of anhydrous N,N-dimercaptoacetamide (DMF) and 4.69 mL of anhydrous under nitrogen. Triethylamine (TEA). The reaction was stirred at 〇 ° C (ice bath) for 8 hrs and quenched by adding 5 mol% excess of EDTA-DA (0.42 mmol, 0.107 g). Stirring was continued for 16 hours at room temperature, and the polymer was precipitated in 1 L of acetone (crude polymer Mw = 144,000 g/mol, GPC, DMAc, PS). The supernatant was decanted; the polymer was rinsed with acetone, air dried, then resuspended in methanol and precipitated in acidified water pH = 2 (HCl). The supernatant was decanted and washed with deionized (DI) water. The collected polymer was then dried under vacuum at room temperature to constant weight. The yield of the recovered product (formula la) was about 60%. The final product after the acid treatment had Mw = 50,700 g/mol (GPC, DMAc, PS) and a glass transition temperature Tg = 77 °C. To achieve higher solubility in water, the prepared polymer was converted to PEA EDTA-Leu by dissolving 5 g of the polymer in 100 mL of NaHC03 saturated solution and dialyzed against deionized water (MWCO = 3.5 KDa). 6) Sodium salt. The lyophilized polymer in the form of a white fluffy powder was recovered in about 50% yield and characterized by 1 H-NMR (Figure 1). Elemental analysis: C28H46N4Na2O10 (644.67); Calculated: C 49.03 s Η 7.83, N 8.27. Experimental values: C 52.17, Η 7.19, Ν 8.69. Mw = 106,000 g/mol, Mw / Mn = 1.42 (size exclusion chromatography (SEC) 10 mM PBS, pH 8.4, OEG standard); Tg = 146 °C. Polymer Synthesis of PEA DTPA-Phe(DAS) (Formula lb): 140309.doc -39- 201039844

COOH

I

Ο H O-C-C-NH- I CH2 〇 ch2 o ho -C-CH2-N-(CH2)2-N-(CH2)2-N-CH2-C-NH-C-C-〇i

Ch2 ch2 ?h2

COOH

COOH Formula 1 is a solution polycondensation, L-Phe(DAS).2 TosOH (18.80 mmol, 14.757 g) is mixed with DTPA-DA (18.80 mmol, 6.718 g), then 15.67 mL of anhydrous DMSO is added under argon and 11.0 mL of triethylamine (TEA). The reaction was stirred at room temperature for 24 hours and the polymer product was precipitated in 2.5 L of acetone. The supernatant was decanted and the polymer was rinsed with acetone and then allowed to air dry. The polymer of formula lb was resuspended in DMSO, diluted with 1:1 v/v deionized water, transferred to a dialysis bag (MWCO = 3.5 K) and dialyzed in deionized water. The dialyzed sample was lyophilized to obtain a white polymer powder of about 90% yield. The weight average Mw = 24,500 (g/mol) (GPC), Tg = 122 〇C. Polymer Synthesis of PEA DTPA-Gly(THF) (Formula Ic): β 〇 d ^o-c-ch2-nh

COOH

I Ο CH2 ο ο C-CH2-*N-(CH2)2-N-(CH2)2-N-CH2-C-NH-CH2C-ch2 CH2

COOH COOH Formula Ic For the polycondensation reaction, Gly(THF)-2TosOH monomer (26.06 mmol, 14.664 g) and DTPA-DA (26.06 mmol, 9.313 g) in 2 1.72 mL of anhydrous dimethyl under argon at room temperature. Mix in yttrium (DMSO) and add 15.26 mL of triethylamine (TEA). The polycondensation reaction was allowed to continue for 26 hours and the polymer product was precipitated in 2.5 L of propylene. The supernatant was decanted and the polymer was rinsed with acetone 140309.doc • 40· 201039844 and then allowed to air dry. The polymer was resuspended in distillation ,2〇, the solution was transferred to a dialysis bag (MWCO=3.5 Κ) and dialyzed for 3 days (deionized water) in distilled Ηβ to be dried to obtain about 50% yield. The white powder material was then characterized by H-NMR and SEC to characterize the product of formula Ic. Mw = 14, 4 〇〇 g / mol, Mw / Mn = 1 62 (SEC, 1 mM mM pBS, pH 8 *, OEG standard). Polymer Synthesis of PEA EDTA-Arg(6) (Form Id): Ο ο ο Η Ο Ο Η

C-CH2-N-(CH2)2-N-CH2-C-NHC-C-0-(CH2)6-〇-CC-NH I CH2 coo- CH2 coo· (CH2)3 NH c=nh2+ NH, (CH2)3 NH C=NH2+ nh2 Formula Id Ο Similar to the preparation of the formulas la, lb and Ic, the polycondensation reaction was carried out at 45 ° C for 1 hour. Then ' increase the temperature to 65. (: further 1 hour to complete the dissolution of the reactants' and then again at 45. (: continued stirring for 6 hours. The polymer was precipitated in the C-class) filtered through filter paper and dried overnight in a vacuum oven. The polymer was redissolved in water together with NaHC〇3 (per 5 g of polymer 〇·5 g of bicarbonate), dialyzed in deionized water for 3 days and dried on a lyophilizer. In the ih_nmr analysis of the polymer of formula Id No counter-p-benzenesulfonic acid counter ion was detected. Elemental analysis: C28H52N1()01() (688.77); Calculated value: C 48.83, Η 7.61 ' Ν 20.34. Experimental value: C 44.95, Η 7·79, Ν 18.76 Mw=17,8〇〇g/m〇l(SEC). The Mw of the polymer was characterized by size exclusion chromatography (SEC).

Waters 600 LC pump, Waters 717 plus autosampler and Waters 140309.doc -41 · 201039844 410 refractive index detector, internal temperature set to 3〇t. A 50 μl aliquot of the sample solution was injected onto a Waters, uhrahydr〇gei (& 7.8 x 3 mm column), which was maintained at 3 (TC and using 1 mM mM ammonium acetate buffer solution ( The pH value was 48) and it was dissolved at 〇6 mL/min. The 2 〇mg/mL PEA-EDTA-Arg(6) polymer sample was dissolved in 1 mM mM ammonium acetate buffer (pH 4.8). The retention time of the substance is a mixture of human thyroglobulin (660 kDa), burdock globulin (158 kDa), ovalbumin (45 kDa), myoglobin (17.8 kDa) and uridine (〇48 kDa). Comparison of the retention time obtained by the protein standard (Phenomonex, Aqueous SEC 1). Synthesis of PEA EDTA Leu (PEG200) of formula Ie: 广?/ X 9 〇〇HN-CH-Ct-〇-CH2_CH2T〇_C-CH -NH-C-CH2-N-CH2-CH2-N-CH2-64-CH2V; k 9H2 ch2 ch2 CH(CH3)2 CH(CH3)2 cooh cooh Jn Formula (Ie) will be double-(L-white Amidino)-PEG2〇〇-diester diterpene sulfonate (L_Leu(PEG2〇〇).2TosOH) (3.177 g) was mixed with EDTA-DA (1.0321 g), followed by addition under nitrogen 2 · 12 mL of anhydrous N,N-dimercaptoacetamide (DMF) and 1.24 mL of anhydrous Ethylamine (TEA). The reaction was stirred at 0 ° C (ice bath) for 6 hours, at room temperature for another 18 hours and quenched by the addition of EDTA-DA (0.26 g). Hour, and the polymer was allowed to stand in 1 L of C. The product was again washed with C-flushing to 'air dry' and then redissolved in 10 mL of saturated NaHC〇3, diluted with 20 mL of deionized water, and deionized. Water dialysis (MWCO = 3.5 KDa). Recovery of 2 g yield cold 140309.doc -42- 201039844 Freeze-dried polymer in white fluffy powder and characterized by 4-NMR (D20, ppm, δ): 0.89 [d , d 12H, -CH-(C//3) 2], 1.60 [m, 4H, -CH-C//2-CH-(CH3)2], 1.74 [m, 2H, -C//-( CH3)2], 2.86 [s, 4H, -NC^-C^-N-], 3.28 [s, 4H, -NH-CO-Cif2-N<], 3.43 . [s,4H, >NC/ /2-COOH], 3.70-3.78 [m,~14H,-0-C/i2-C^- 0-], 4.26-4.33 [m,m, 4H, -0C0-C//2-CH2-0 -], 4.47 [m, 2H, -HN-C/i<]. Mw = 33,000 g/mo b Mw/Mn = 1,04; (SEC, 10 mM PBS pH 8.4, +20% v/v MeOH, OEG standard).制备 Preparation of polymer metal conjugates and determination of binding capacity Water-soluble polymer PEA-DTPA-Leu(6) is mismatched with Gd(Ul): chromium mg PEA-DTPA-Leu(6)-Na salt (Mw 13,100 g/mol, GPC, DMAc, PS) was dissolved in about 8 mL of deionized water. Then, an equimolar amount of an aqueous solution of GdCl3.6H20 was added dropwise to the solution under stirring. The pH was maintained at 5.8 by the addition of 0.1 M NaOH. Stirring was continued for 1 day. Free Gd ions are no longer detected by dialysis of the solution into the solution (dioxophenol orange test, as described by Barge, ❹ A. et al. Ccmirasi Mo/. (2006) 1:184-188), followed by lyophilization of the sample. . A decrease in the apparent molecular weight of the polymer (Mw = 8,700 g/mol) was observed after the mismatch, which was attributed to the charge neutralization in the DTPA polymer when bound to the metal. Metal bonding further causes a change in the hydrodynamic value. According to the ICP-MS measurement, the content of Gd(III) per DTPA cage was > 90%. Example 2 PEA synthesis based on EGTA [CO-EGTA]: - Dad reaction (flow 1) 140309.doc -43- 201039844

Wide COOH (CF3C0)20 J > V-COOH 〇-5°C bis(L-Leu)-l j-hexanediol.2TosOH COOH I ch2 C-CH2-N-(CH2)2〇-(CH2) 2-〇-(CH2)2-N-CH2

Ch2 COOH

Procedure 1 30 mL of anhydrous dichlorohydrazine (DCM) and 5 g (13.1 mmol) of ethylene glycol-bis(2-aminoethyl ether tetraacetic acid (EGTA) were charged into a 250 mL 3-neck round bottom flask. It was cooled on an ice bath and covered with argon. Next, 4.55 mL (33 mmol) of trifluoroacetic anhydride was added and stirred until the white solid was completely converted to a clear pale yellow EGTA dianhydride viscous layer (about 4 hours). The ice bath was replaced with a methanol/dry ice bath and the reaction mixture was cooled to -4 ° C to -30. (: I6.5 mL (0.118 mol) of triethylamine (TEA) was separately diluted in 20 mL of anhydrous DMF. And added dropwise to the reaction mixture over a period of 1 hour, and stirring was continued for 30 minutes at about -30 ° C. Then 9.040 g (13.1 mmol) of bis-(L-leucine)-l,6-hexane was added. The diamine monomer diamine monomer di-p-benzoate and was stirred overnight at room temperature. The crude polymer solution had Mw = 3 6 kg / mol, Mw / Mn = 1.462 (GPC, DMAc, PS). The reaction solution was rotary evaporated to remove volatile DCM, diluted with 20 mL of water and dialyzed against deionized water. After lyophilization, 5.94 g of polymer 'Mw = 3 〇 kg/m 〇 USEC, PEO) was collected. /acetic acid B Further purification of the polymer by ester reprecipitation. The polymer structure of the invention was confirmed by 1H NMR analysis in D20. 140309.doc -44· 201039844 Example 3 PEA.EDTA.Leu(6). Nickel [Paclitaxel] Nanoparticles This experiment was carried out to illustrate the procedure of the present invention for formulating the metal chelate polymer of the present invention into nanoparticle to deliver the water-insoluble bioactive agent paclitaxel.

潆合勿潆竑潆竑旋之农潆: 120 mg of the polymer of the invention PEA.EDTA.Le\i(6) at room temperature (Mw=24 kg/mol, 〇Mw/Mn=1.68 Formula I, wherein RL-CHrNCCI^COjHKCHzhN (ch2co2h)-ch2-; R3=CH2CH(CH3)2, R4=(CH2)6) is dissolved in 3 mL of 1-methyl-2-n ratio (NMP) And, at a rate of 1 mL/min, was added dropwise to 17 mL of 25 mM N-(2-pyridylethyl)nazine-N'-(2-ethanelithic acid) (HEPES) aqueous buffer. Liquid (pH = 7.0). The buffer solution was vigorously stirred at room temperature to produce a homogeneous polymer solution having a concentration of 6 mg/mL. Stirring was continued for 15 minutes and then the solution was dialyzed against 2 l of 25 mM HEPES buffer (ρ Η = 8.0) containing 15 mM mM NaCl overnight. The dialysis membrane is a mixed cellulose (SpectroporeTM) having a molecular weight cut off 〇 (MWCO) of 12-14 kDa. The final polymer recovery after dialysis was estimated to be 82% as estimated by amino acid analysis. Amino acid analysis was carried out by hydrolyzing the polymer in 6 N hydrochloric acid under an inert atmosphere. The hydrolyzate was then derivatized with fluoromethane carboxylic acid 6-aminoquinolinyl-•N-pyridinium succinimide ester and then analyzed by reverse phase HPLC. Preparation of paclitaxel/NiCl2 stock solution (B): A solution of 2 mg of paclitaxel (ρτχ, LC Labs) in 0.95 mL of NMP was prepared by mixing with mash. 5 · 16 mg NiCl 2 (Sigma) was dissolved in 0.2 mL of deionized water in separate vials. A PTX/NiCl2 stock solution (B) was prepared by adding 0.05 mL of a NiCh aqueous solution to 0.95 mL of PTX solution 140309.doc 45·201039844 by rapid addition, which contained 2 mg/mL PTX and 1.29 mg/mL. NiCl2 (95% v/v NMP and 5% v/v H2〇). The mixture was stirred by vortexing and designated as phase (C). 3.1 PEA.EDTA.Leu(6)Ni [PTX] nanoparticle of the invention preparation method: 0.5 mL of PEA.EDTA.Leu (6) stock solution (A) diluted with 2_5 mL 25 mM HEPES to produce 0.1% polymerization An aqueous solution, designated as phase (D). PEA.EDTA.Leu(6)Ni [PTX] Nai was produced during the drop at room temperature during the addition of 0.25 mL phase (C) to the 3 mL phase (D) at an addition rate of 0.25 mL/min. Rice particles. The mixture was stirred for a further 5 minutes and dialyzed against 2 L of 25 mM HEPES (pH = 7.0) overnight. The dialysis membrane is a mixed cellulose (SpectroporeTM) having a MWCO of 12-14 kDa. The monomodal z-average diameter of the formed nanoparticle dispersion was measured by a dynamic light scattering (Malvern Zetasizer) of 151.1 nm, and the mean zeta potential was -45.5 mV. The final PTX recovery of the nanoparticles was 56.5 pg/mL as determined by HPLC (ACN/H20 USP method), and the final polymer recovery was 54% as determined by the amino acid analysis. 3.2 # I^PEA MM PEA.EDTA.Leu(6)Ni [PTX] # ^ In the formation of the sub-method: For the purpose of comparison, repeat the above description of the formation of product nanoparticle in Section 2.1 Procedure, but replace with 0.5 mL of PEA stock solution with only 500 μl of 25 mm HEPES. Using this procedure, a crystal aggregate having a size of 3 to 300 μηη is formed as determined by optical microscopy using a hemocytometer. 3.SM !^NiCl2^PEA.EDTA.Leu(6)Ni [PTX] ^ 140309.doc -46- 201039844 The original distribution method: repeat the above description of the formation of product nanoparticles in Section 2.1 Procedure, but replace with 50 pL NiCl2 stock solution to add 50 μί deionized H20 to PTX in 0.95 mL NMP. The result after dialysis is the formation of a crystalline agglomerate having a size in the range of 10 to 500 μη. Example 4 PEA.EDTA.Leu(6). Nickel [ΡΤΧ]-[6-Histidine-labeled Green Fluorescent Protein] Preparation of Nanoparticles _ This experiment was conducted to illustrate the formulation of the nickel chelate polymer of the present invention. The present invention is a process for simultaneously delivering a hydrophobic bioactive agent and a His-tagged target protein (such as an antibody or other known proteinaceous targeting ligand) to a water-soluble quinone target nanoparticle. 6His-labeled GFP, which is a protein rather than a peptide, was used to mimic the procedure for chelation of a His-tagged target protein with a metal chelate polymer of the invention to deliver the highly hydrophobic drug paclitaxel.衮合#锗潆竑荔放之I Μ吏 Dissolve 40 mg of free acid form of PEA EDTA-Leu(6) (Mw=25 kDa, p Mw/Mn=1.59, GPC, PS) in an ultrasonic bath In 4 mL of 25 mM HEPES buffer (pH = 11.2). The pH after complete dissolution was 7.4. The target PEA concentration was 10 mg/mL as determined by amino acid analysis and the final polymer recovery was 92%. Preparation of PTx./mci2 stock solution (b): At % 1 fly m a % mg PTX was dissolved in 967 μί NMP. In a separate container, dissolve the 5.16 1^ (^12(8丨8111&) in 0.2 mL of deionized water at room temperature using a vortex mixing and ultrasonic bath. Quickly add to the 967 μί PTX solution. A 33 μί NiCl 2 aqueous solution produces a stock solution of PTX/NiCl 2 (Β). The mixture is stirred at 140309.doc -47- 201039844 and will contain 〇·68 mg/mL paclitaxel and 0.85 mg/mL NiCl2 (97% v/v NMP and The final stock solution of 3% v/v H20) is designated as phase (C) PEA.EDTA.Leu(6).Ni [PTX]-[6-histidine-labeled green fluorescent protein (6His-GFP)] Nanoparticles of the invention are prepared by diluting 0.1 mL of PEA.EDTA.Leu(6) stock solution (A) with 3.4 mL of 25 mM HEPES (ρΗ=7.0). Add 0.5 mL of Tris buffer physiology as a bolus. 1 mg of 6His-GFP in saline (TBS, pH = 7.0). The homogenous mixture formed as phase (D) was stirred for a further 5 minutes at room temperature by magnetic stirring at room temperature. 0.25 mL/min addition rate 0.25 mL phase (C) was added dropwise to phase (D) to produce PEA.EDTA.Leu(6)Ni [PTX]-[6His-GFP] nanoparticles. Stirring continued for 5 Minutes and mixture The solution was dialyzed against 500 mL of 25 mM HEPES (pH = 7.0) overnight in a mixed-fiber (SpectroporeTM) membrane with a MWCO of 12-14 kDa. The nanoparticle dispersion after dialysis has 86 11 as determined by dynamic light scattering (Malvern Zetasizer). «1 of 2 average diameter, and -3 7.4 111¥ of 6 potential average. As determined by 11?1^ (ACN/H20 USP method), the final PTX recovery of nanoparticle is 14.9 pg/mL, and finally The polymer recovery was 96% as determined by amino acid analysis. The final 6His-GFP recovery in nanoparticle was 49 as measured by GFP fluorescence (Fluostar Optima) emitted at 520 as excited at 485. Example 5. PEA.EDTA.Leu(6). Nickel [PTXH Bovine Serum Albumin (BSA)] Preparation of Nanoparticles 140309.doc -48· 201039844 This experiment was conducted to illustrate the formulation of the metal chelate polymer of the present invention. For the nanoparticle to pass the bioactive agent paclitaxel by the common blood protein (bovine serum albumin) target. 衮合#锗潆竑荔放之农潆: 150 11^ free acid form by ultrasonic treatment bath ? £八』0丁八.1^11(6)(汲汲=25]^3,1^\¥/]^11= 1.59 ' GPC ' PS) Dissolved in 15 mL 25 mM HEPES buffer (ρΗ=11 · 15) Medium. After complete dissolution, the final solution designated as solution (a) Ο PH value: 4. As measured by amino acid analysis, the concentration of PEA was 1〇 111§/1111^, and the final polymer recovery was 83%. PEA.EDTA.Leu(6)Ni[PTX]-[Bovine Serum Albumin (BSA)] Nanoparticles I 涝·· 0.1 mL of PEA.EDTA_Leu(6) stock solution (A) with 3.8 mL 25 mM HEPES (pH = 7.0) diluted and mixed with 1 mg BSA (Fraction V, Sigma) in a 0.1 mL 25 mM HEPES (pH = 7.0) solution. The homogeneous mixture formed as phase (B) was stirred at room temperature for 5 minutes. Next, while disturbing at room temperature, a phase (C) prepared by adding 250 μl of the phase (C) prepared as described in Example 4 above was added dropwise to the phase (B) (addition rate 0.25 mL/min). A dispersion of PEA.EDTA.Leu(6)Ni [PTX]-[BSA] nanoparticles was produced. The dispersion was dialyzed against 0.5 L of 25 mM HEPES (pH = 7.0) overnight in a mixed cellulose (12 - 12 kDa) mixed cellulose (SpectroporeTM). After dialysis, the dispersion was measured by a dynamic light scattering (Malvern Zetasizer) with a single mode z average diameter of 65.7 nm and an average of the zeta potential of -29.2 mV. The final paclitaxel recovery of the nanoparticles was 19.6 pg/mL as determined by HPLC (ACN/H20 USP method), and the final polymer recovery was 73% as determined by amino acid analysis. The final BSA recovery in the nanoparticles 140309.doc -49 - 201039844 was 73% as determined by amino acid analysis. Example 6 PEA.EDTA.Leu(6) Zinc [6-Histidine-labeled Green Glory Protein] Formulation of Nanoparticles This experiment was carried out to illustrate the formulation of a zinc chelate polymer as a nanoparticle to incorporate a His tag. The inventive procedure for proteins. Recording the field record #i落放64) 裘7 Guang: In the ultrasonic bath, 22.6 mg free acid form of PEA.EDTA.Leu(6) (Mw=34 kDa, Mw/Mn=1.67 ' GPC , PS) was dissolved in 2.26 mL of 25 mM HEPES buffer (ρΗ=7·0). The final solution pH was 7 〇. The final concentration of pea was 10 mg/mL, which was designated as stock solution (a).

Ning #彦淡(8) Attack 7 Infiltration 100 mg of ZnCl2 was dissolved in 50 mL of 25 mM HEPES buffer (pH 7). The concentration of ZnCl2 stock solution was 2 mg/mL. When 1.06 mL of the ZnCl2 stock solution (b) was added to 3.94 mL of HEPES (pH 7.0), ZnCl2 was designated as the final concentration of the solution (B) of 0.423 mg/mL. 6.1 PEA.EDTA.Leu(6) Zn-[6His-GFP] Nanoparticles (C): Preparation of 850 pL PEA.EDTA.Leu(6) stock solution (A) at 7.65 mL 25 mM HEPES (pH The dilution in =7.0) to produce a polymer concentration of 1 mg/mL. Add 1 mg of 6His-GFP from 1 mL of Tris-buffered saline (tbs, ρΗ=7·0) as a bolus to 2 mL of diluted sputum stock solution (Α) and homogenize the mixture at room temperature Stir for 5 minutes. The PEA.EDTA.Leu(6)Zn-[6His-GFP] nano 140309 was produced by adding 1 mL of ZnCl2 solution (B) dropwise at a rate of 0.25 mL/min under magnetic stirring at room temperature. Doc -50- 201039844 Particles. The mixture was stirred for a further 30 minutes. The nanoparticles formed in the dispersion (6.1) had a z-average diameter of 31 nm as measured by dynamic light scattering (Malvern Zetasizer). The final 6His-GFP recovery in the nanoparticles was 84% as measured by GFP fluorescence (Fluostar Optima) emitted at 520 as excited at 485. 6.2 PEA.EDTA.Leu(6) Zn-[6His-GFP] non-PEA control formulation of agricultural carp, repeat the above procedure for preparing the formulation (6.1), but omit 2 mL PEA solution (A) and replace with 2 mL 25 mm HEPES (pH 7.0). The resulting formulation was determined to contain crystalline aggregates without nanoparticle. This experiment shows that the metal chelate polymer of the present invention must be present to obtain nanoparticle using a polycondensation method. 6.3 PEA.EDTA.Leu(6) Zn-[6His-GFP] Nanoparticles of non-ZnCl2 Shore Original Deer #裘潆: Repeat the procedure for preparing the formulation (6.1), but omit 1 mL of ZnCl2 solution and Replace with 1 mL of 25 mM HEPES (pH 7.0). The resulting dispersion (6.3) has a particle diameter in the range of 9 to 500 nm. This experiment shows that the presence of metal ions in the polycondensation procedure helps to form nanoparticles prepared using the polycondensation process of the present invention. Example 7 PEA.EDTA.Leu(6). Nickel [PTX1-[bovine serum albumin (BSA)] nanoparticle blending compound #锗#竑溶旋)): 87 mg by vortex stirring PEA EDTA-Leu (6) (Mw = 82 kDa, Mw / Mn = 1.23, (SEC)) in the form of the sodium salt was dissolved in 8.7 mL of 25 mM HEPES buffer (ρΗ = 7.0). After dissolution, the sample was filtered through a 0.45 μη GHP (hydrophilic polypropylene) disc filter (Pall Life Sciences). The final pH after filtration was 7.54. For example, 140309.doc -51 - 201039844 Amino acid analysis estimated that the final polymer recovery was 79 8%. Preparation of PTX/NiCh stock solution (B): 15 mg of sputum tower was dissolved in 750 Torr. In a separate container, 4.02 mg of NiCl2 (Sigma) was dissolved in 〇 25 mL of deionized water by vortex stirring and ultrasonic bath at room temperature. A stock solution (B) of PTX/NiCh was produced by rapidly adding 25 Torr of pL NiCh aqueous solution to 750 pL of PTX solution. Mixture

The final stock solution of 7.5 mg/mL paclitaxel and 4.02 mg/m]L v/v NMP and 25% Wv out of 0) was designated as phase (c) by vortexing. PEA EDTA-Leu(6)Ni [PTX]-[bovine serum albumin (BSA)] nanoparticle 裘;# .· 2.0 mL PEA.EDTA.Leu(6) stock solution (A) to 6 mL 25 Diluted with mM HEPES (pH = 7.0) and mixed with 20 mg BSA (Fraction V, Sigma) in 1.0 mL 25 mM HEPES solution (pH = 7. 。). The homogeneous mixture formed as phase (D) was stirred at room temperature for 5 minutes. While stirring at room temperature, the 1 〇〇〇 phase (C) was added dropwise to the 9 mL phase (D) at an addition rate of 0.25 mL/min to produce PEA.EDTA.Leu (6)Ni [ PTX]-[BSA] nanoparticle. The nanoparticle dispersion was dialyzed overnight (16 hours) against 0.5 L of 25 mM HEPES (pH = 7.0) in mixed cellulose (SpectroporeTM) having a MWCO of 12-14 kDa. After HEPES dialysis, the samples were further dialyzed against 〇.5 L 0.9% NaCl (VWR) for 3 hours in a similar dialysis tube. The nanoparticle in the dispersion after dialysis was measured by a dynamic light scattering (Malvern Zetasizer) having a z-average diameter of 118.3 nm and an average value of the zeta potential of -17 mV. The final paclitaxel recovery of the particles was 668 pg/mL as determined by HPLC (ACN/H20 USP method), and the final polymer recovery was 67% as determined by amino acid analysis. The final BSA recovery was 74% as determined by the analysis of the amino acid group 140309.doc -52- 201039844. These results are summarized in Table 5 below. Table 5 The molar ratio of paclitaxel to BS A in paclitaxel Mw=853.9 g/mol BSAMw=66,430 g/mol in Example 7. BSA theoretical mass: 20_0 mg BSA theoretical molars: 0.301 μπιοί BSA experimental quality (via AAA): 14.8 Mg O BSA number of moles (from AAA mass): 0.223 μπιοί Paclitaxel theoretical mass: 7.5 mg paclitaxel theoretical molars: 8.78 μπιοί paclitaxel experimental mass (by HPLC): 6.68 mg paclitaxel experimental moles (from HPLC quality) : 7.82μπιο1 Theory Paclitaxel: BSA Mo Erbi: Mohr ratio of paclitaxel to BSA (8.78 μηιο1/0.301 μηιο1)=29.2 Experimental Paclitaxel: BSA Mo Erbi: Q Molar ratio of paclitaxel to BSA (7.82 μιηο1/0.223 μιη〇 1) = 35 Example 8 The chelating polymer of the present invention (e.g., PEA EDTA-Leu (6) (formula la)) is soluble in aqueous solution and thus provides a benign environment for the formulation of sensitive biomolecules. Molecules are structurally unstable in organic environments such as nucleic acids (including RNA), antibody fragments, protein domains, and whole proteins. The ability of the polymer to induce condensation using a metal allows the formulation component to be captured in the nanoparticle or microparticles as well as in the protein present on the surface of the particle. This latter feature is particularly useful for formulating putative vaccine antigens for testing. Recombinant technology available 140309.doc -53- 201039844 in the sequence of these protein antigens

a j γ is added to the poly(histidine) segment, which is the "H tag". The His-tagged protein temporarily promotes the binding of the antigen to the chelated human polymer via the metal ion, and when the formulation is administered in the form of Λ Λ, the natural folding antigen site is presented to the immune system. The His-tagged polypeptide for use in combination with the chelated polymer milk of the present invention (such as 'PEA EDTA-Leu(6)) can be produced from any known expression system, such as mammalian tissue. Culture, baculovirus infection, insect cells, yeast and bacteria. Typical protein purification involves the solubilization of cells by microfluidization followed by ion exchange chromatography and immobilized metal affinity chromatography (IMAC). Proteins prepared for use as vaccines against infectious diseases such as influenza should preserve the naturally folded protein domain and thus induce humoral and cellular immunity from the immune system of the individual receiving the vaccine. A formulation of a His-tagged protein prepared using the chelate polymer of the present invention and a method can be prepared to incorporate one or more proteins into the polymer particles and then the formulation can be mixed, or the vaccine particles can have or Individual administration without additional additives such as adjuvants or dry-facing fractions. Because the naturally occurring conformational state of the influenza virus hemagglutinin (HA) is critical for a robust protective B cell response, and protection can be provided by antibodies directed against all parts of the viral protein, baculovirus infection A metal condensate formulation of PEA EDTA_Leu (6) with a portion of the influenza virus HA protein naturally exposed on the viral surface (extracellular domain) is produced in SF9 cells. SF9 cells at a density of 1.5 x 1 〇6 cells per ml were infected with 500 g of Sf900 II-SFM medium (Invitrogen, San Diego CA) using a multiplicity of infection (M 〇 I = 1) of pBac_HAPR8 baculovirus. The infected 140309.doc -54· 201039844 cells were grown for 48 to 72 hours and collected by centrifugation. Immobilized metal affinity chromatography by solubilization of cell proteins by suspension in Pbs buffer containing 0.1 ° / 〇 Triton X-100 and protease inhibitors and then by using Ni-loaded chelated agarose (GE) IMAC) purified it. The purified protein was dialyzed against 50 volumes of 25 mM Tris (pH 8.0), 150 mM NaCl, and replaced twice and filtered through a 2 micron filter. Since hemagglutinin antigens need to preserve their natural folding for the purpose of utility, follow standard protocols (ie, Webster, R; Cox, N and Stohr, K, WHO Animal Ο

Influenza Manual, World Health Organization, WHO/CDS/NCS/2002.5) tests the sialic acid binding function of the recombinant HA-derived domain by hemagglutination assay. Chicken red blood cells were used in agglutination assays using A/Puerto Rico/8/34 influenza virus as a control.

The DNA sequence (NPPR8, SEQ ID ΝΟ: 1) encoding the nucleoprotein (NP) from influenza A/Puerto Rico/8/34 was designed to encode amino acid 1 to 498 (Genebank accession number NP_040982) plus six His label. The sequence (NPVN, SEQ ID NO: 5) from NP of influenza A A/Vietnam/1203/2004 encodes amino acid 1 to 495 (Genebank accession number AAW80720) plus a six His tag. The carboxy-terminal hexahistamine is included in the gene cassette encoding each of these viral NP sequences to aid in purification and polymer loading. The influenza nucleoprotein (NP) gene cassette was prepared by overlapping oligonucleotides and PCR synthesis and was subsequently cloned into pET26b (Novagen). The NPPR8 and NPVN expression vectors were transformed into BL21-DE3. Bacteria were grown to saturation in selective TB medium (Genessee Scientific) and then diluted twice with fresh ice-cold medium of 140309.doc -55-201039844. Protein expression was induced with 200 μΜ IPTG in these cultures at room temperature. After 4 to 6 hours of induction, the bacteria were centrifuged and the obtained centrifuge pieces were frozen. The prion protein was purified by IMAC. The bacterial pellet was thawed in phosphate buffered saline (ΡΗ 7.4) (PBS) and solubilized by ultrasonic treatment. The bacterial lysate was centrifuged at 23,000 xg and the supernatant was adjusted to 25 mM. Sit' then pass through a pre-loaded chelating Glucose column (GE Healthcare). The loaded column was sequentially subjected to 50 column volumes of ice-cold wash buffer 3 (50 mM sputum, 150 mM NaCl '0.1% Triton X-114, 25 mM sodium phosphate, pH 7.5) and 20 column Wash the volume of Wash Buffer 4 (5 mM mM imidazole, 150 mM NaCl, 25 mM sodium phosphate 'pH 7.5). The HA protein bound to the column was detached with 500 mM imidazole in PBS. The solubilized np protein was dialyzed against two volumes of PBS loaded twice. The endotoxin content of purified recombinant NPPR8 and NPVN was routinely tested by the chromogenic cell lysate (LAL) assay (Cambrex) and used using known methods (Reichelt, P.5 C. Schwarz and M. Donzeau, "Single step protocol to purify recombinant proteins with low endotoxin contents." Pro, such as "five wr wr / / (2006) 46 (2): 483-8) with additional IMAC cycle of Triton Xl 14 washing j solution and then purified The protein solution contains less than 1 endotoxin per ml. A blend of PEA EDTA-Leu (6) and His-tagged purified recombinant influenza protein was prepared as follows. The acetic acid is sedated in citrate physiological saline

The solution in the flush (pH 7) was slowly dropped into the hexa-His-labeled HAPR8 extracellular domain (SEQ ID 140309.doc -56 - 201039844 NO: 2) and 25 in 25 mM Tris, 150 mM NaCl (pH 8). In a stirred mixture of PEA-EDTA-Leu (6) in mM HEPES (pH 8), a His-tagged HAPR8 extracellular domain (SEQ ID NO: 2) with a final concentration of 1 mg/mL was produced, 1.5 mg/mL PEA -EDTA-Leu(6) and .367 mg/mL acetic acid. The His-tagged NPPR8 (SEQ ID NO: 1) formulation was prepared using the same procedure, but the Npprs protein was introduced into 25 mM sodium citrate, 150 mM NaCl (pH 7). The NPPR8-Zn-EDTA-Leu(6) formulation contained a final concentration of 〇465 mg/mL His-labeled NPPR8 (SEQ ID NO: 1), 0.233 mg/mL PEA-EDTA-Leu (6), and 0.05 7 mg/ mL acetic acid. The metal ion condensate of the PEA chelating polymer and the influenza antigen was conventionally stored at 4 ° C until administration. PEA EDTA-Leu(6)-Zn-influenza protein antigen test was performed by administration to B6/C3 F1 mice. The humoral responses of these animals to HA and NP antigens were assessed using quantitative ELISA by assessing antibodies produced in serum and broncho-alveolar lavage fluid. The sputum cell response was assessed by measuring the interferon gamma by ELISPOT. Interferon gamma production was assessed by spleen cells isolated from immunized mice that had been re-stimulated from HA or sputum peptides. Figures 2 and 3 show data from experiments in which a dose of PEA-EDTA-Leu (6) containing 25 pg of HAPR8-3 and 9 pg of NPPR8 was administered intranasally to mice. The mice were bled on day 14 and challenged intranasally with 10 LD50 infectious virus on day 21. Animal morbidity and mortality were monitored over the next three weeks. In Figure 2, the data show that animals administered a separate dose of influenza protein formulated with zinc and PEA EDTA-Leu (6) did not survive unless the formulation also contained the adjuvant poly I:C. Despite significant body weight loss in these surviving animals (Figure Π), small 140309.doc •57-201039844 rats receiving formulations containing poly-I:C adjuvant survived viral challenge after only one vaccine of the invention was administered . This survival was correlated with ELISA data. The data showed that only the intraperitoneal virus-administered group and the HAPR8 extracellular domain and NPPR8 formulated with PEA EDTA-Leu(6)-Zn with poly-I:C adjuvant were used. The group of animals produced an anti-HA IgG2a antibody at a level of 100 ng/mL or more than 100 ng/mL. All animals that received a formulation containing the formulated NPPR8 protein produced high levels of anti-NP antibodies. Example 9 In this study, the hemagglutinin (HA) domain produced by the baculovirus having agglutination ability and the globulin lectin (ha) domain produced by the bacteria were used as putative influenza antigens. The hemagglutination assay described above was used in conjunction with an agglutination inhibition assay to assess formulation candidates. If the hA protein or protein subdomain tested has dry binding activity prior to formulation into the vaccine of the invention, the His-standard HA that is formulated with a cation (such as Zn2+, Mn2+ or Ni2+) must also have hemagglutination. active. Depending on the organism used for manufacture, examples of influenza hemagglutinin antigen fragments (SEq ID Ν〇 2, 3, 4, 6, 7, 8) or similar sequence fragments from other influenza prion proteins It may be manifested with or without a bacterial signal sequence (which is underlined in SEQ ID ΝΟ: 3, 4, 7 and 8). The purified protein tested by this hemagglutination test can be used as a good influenza antigen. The muscle influenza vaccine has also been tested, in which all of the protein components of the successful vaccine PEA EDTA_Leu(6)-Zn formulation are purified from bacteria. In the immunoassay described below, the formulation was supplemented with poly-I:c as an adjuvant, and the formulation contained an additional H0309.doc •58·201039844 NPPR8 compared to the vaccine candidates described in the sputum example. In addition, this research test is dedicated to eliminating the initial-boost regimen of vaccinated morbidity after infection. Preparation of PEA EDTA-Leu (6) (formula la) and His-tagged HA polypeptide of bacterial expression as described in Example 8 (for example, HAPR8-3 (SEQ ID NO: 4) or 11 VIII \^-3 (8 ugly a formulation of (^10>10:8)), but including a bacterial signal sequence in each sequence. The solution of acetic acid in a citric acid physiological saline buffer (pH 7) is slowly dropped into the tris a stirred mixture of His-labeled HA polypeptide in physiological saline solution (pH 8) and PEA EDTA-Leu (6) in citrate physiological saline buffer (pH 7), which is sufficient to produce The final concentration of 1_1 mg/mL His-tagged HA polypeptide, 0.55 mg/mL ugly-£0-in-1^11(6) and 0.12〇11^/111]^ zinc acetate. As in Example 8 Prepare a NPPR8 (SEQ ID ΝΟ:1) formulation for use with a His-tagged HA polypeptide formulation of bacterial expression, but with a final concentration of 1_1 mg/mL NPPR8 (SEQ ID ΝΟ: 1), 0.55 mg/mL PEA EDTA -Leu(6) and 0.12 mg/mL zinc acetate. To test the effect of different routes of administration of the PEA-EDTA-Leu(6) particle formulation and the His-labeled influenza antigen, 10 Balb/c Subcutaneous or intranasal administration of the mouse group Compounds. The efficacy of the two routes of administration was then compared. The animals were pre-sensitized with a formulation containing 50 HAPR8-3 and 25 pg NPPR8 at 50 μΐ dose. Each was formulated as a PEA containing 5 pg of poly I:C EDTA-Leu(6)-Zn particles. Two weeks after the first administration, each group of mice was boosted with the same mixture of the second dose. After 3 weeks, all mice were subjected to 10 LD5〇 infectious A/Puerto Intranasal infection of Rico/8/34 virus. The results of this test 140309.doc -59- 201039844 indicate the importance of the route of administration for these microparticle formulations. For intranasal administration with Zn and PEA EDTA-Leu ( 6) - For animals formulated with the HAPR8-3 and NPPR8 proteins, 90% of the animals survived the infectious challenge. Relatively speaking, only 10% of the animals administered the same formulation subcutaneously survived. The degree of weight loss in response to viral infection as described in 4 reflects that the mice given the intranasal vaccine also showed a reduced incidence. These results show that the mice vaccinated intranasally at the same vaccine and dose have a lower than subcutaneous dose. Better immune response to the mice in the vaccine. Example 10 About Flows 2 and 3 below The depicted end group conjugates illustrate the following conjugation strategy: In a first example of end group conjugation, the PEA chelate polymer of the present invention is synthesized primarily with amine end groups and then with a single activated PEG (eg, mPEG- SVA, mPEG-pentyl succinimide, conjugated from Laysan Bio Inc, Arab, AL). The reaction was carried out in an aprotic organic solvent (DMSO, NMP) according to Scheme 2 below. mPEG-linker-OSu H2N-PEA EDTA-Leu(6)-NH2 -- Ο Ο

PEG-linker-C-HN-ΡΕΑ EDTAJ^eu(6)-NH-C-linker-PEG Scheme 2 The anhydride end group in the B polymer used as shown in Scheme 3 below also allows macromolecules via amine or hydroxyl groups. Or the active drug is further mixed to produce a guanamine or ester bond. 140309.doc •60· 201039844

Q N^—PEA EDTA_Leu(6)-

A) mPEG-NH2

0 B) mPEG-OH 0 0 PEG-HN丄"Λ /-^NH-PEG A) N^^—PEA EDTA Leu(6}—H〇tf S-〇H 0 0 0 0 B) PEG—O丄Λ ^-Lo-PEG N-λλ-pea EDTA Leu(6>- Η〇τ f ^ir0H o 0 Scheme 3 PEA EDTA-Leu(6) and dianhydride end synthesis and further conjugation with mPEG-NH2 ΑΒΑ block polymer. 5.218 g (7.6 mmol, 0.91 eq.) of L-Leu(6)-2TosOH, 2.1326 g (8.3 mol, 1.00 eq.) EDTA-DA was suspended in 2.3 mL of anhydrous dimethyl sulfoxide (DMSO). The suspension was covered with argon. Then, 4.64 mL (33 mmol) of triethylamine was added and stirring was continued for 3 hours at room temperature. (Mw=51,500 g was obtained by analyzing the Mw of the crude sample by GPC (DMAc, PS). /mol) Next, 2,01 g of mPEG-amine (Mw 5000, Laysan Bio Inc, Arab, AL) and 4 mL of DMSO were added and stirring was continued overnight at 50 ° C. The polymer was precipitated in 500 mL of acetone, and then Dissolved in 100 mL of deionized water. To completely dissolve the polymer, 15 mg of NaHC03 was added and the solution was dialyzed against deionized water in a MWCO = 12-14 KDa dialysis bag. The yield of 2.2 g was recovered as a white fluffy powder. The lyophilized polymer was confirmed by 1H-NMR (MeOD) to confirm the presence of a total of PEG. Mw = 36,000 g/mol, Mw/Mn = 1.38; (SEC, 10 mM PBS pH 8.4, + 20% v/v MeOH, OEG standard). 140309.doc -61 - 201039844 PEA EDTA-Leu(6)-two liver end polymer and lamina sugar (laminarin) in another example, such as Portuguese The polysaccharide adjuvant of the glycan is conjugated to the end group of the chelate polymer of the present invention. In this example, a representative glucan laminide commercially available is used as a representative polysaccharide adjuvant suitable for vaccine preparation. 4 complete the conjugate of the adjuvant:

〇 昆 昆布糖-0—®\ /—L〇- lenbuxose

_► N~^—PEA EDTA_Leu(6)—^ N Η0_ΙΓ^ νΠΓΟΗ ο ο Scheme 4 More specifically, 4.283 g (6.2 mmol, 0.84 equivalent) L-Leu(6)-2TosOH, 1.8926 g (7.4 mol) 1.00 eq.) EDTA-DA was suspended in 7.95 mL of anhydrous TV-methyl-2-pyrrolidone (NMP) and covered with argon. Next, 1.9 mL (14 mmol) of triethylamine was added and stirring was continued for 16 hours at room temperature. (Mw of the crude sample was analyzed by GPC (DMAc, PS) to give Mw = 5 1,000 g/mol). 1 g of laminarin (Aldrich, Mw = 5,000 g/mol) was separately dissolved in 7.5 mL of NMP and 2 mL of polymer reaction solution (about 2 mL) was added, followed by the addition of an additional 13.9 pL of TEA and the solution at 60 ° C. Stir for another 16 hours. The solution was diluted with 100 mL of deionized water, transferred to a 12-14 KDa MWCO dialysis bag and dialyzed against deionized water. A 1.18 g yield of the lyophilized polymer in the form of a white fluffy powder was recovered. Tested 140309.doc -62- 201039844 The conjugated polymer was negative in the indanone test. The presence of a 37% w/w loaded conjugated laminin was confirmed by W-NMR (DMSO-A). Mw = 70,000 g/mo b Mw/Mn = 1.2; (SEC, 10 mM PBS pH 8.4, +20% v/v MeOH, OEG standard). All publications, patents, and patent documents are hereby incorporated by reference in their entirety herein in their entirety herein The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention. Although the present invention has been described with reference to the above examples, it should be understood that modifications and variations are included within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a 1H-NMR spectrum representation of a polymer: pEA£D7M-i>w(^) (formula la); Figure 2 is a graph showing an infected mouse infected with an influenza virus. Save the graph of the Q curve. Solid ◊ = animal immunized only with buffer; ▲ = animal immunized with virus intraperitoneally, positive control; star = HAPR8 extracellular domain formulated with pea EDTA-Leu(6)-Zn and poly I:C NPPR8 intranasal • immunized animals; = HAPR8 extracellular domain formulated with PEA EDTA-Leu(6)-Zn) and intranasal immunized mice with NPPR8; Figure 3 shows the immunization of immunized mice A graph of changes in body weight after a cold virus infection. 〇 = animals immunized only with buffer; star = animals immunized intraperitoneally with virus, positive control; ▲ = HAPR8 extracellular domain and NPPR8 formulated with PEA EDTA-Leu(6)-Zn and poly I:C Intranasal immunization of animals with average changes in body weight of 140309.doc -63- 201039844; = intracellularly immunized with HAPR8 extracellular domain and NPPR8 formulated with PEA EDTA-Leu(6)-Zn-; PEA EDTA-Leu(6)-Zn - an animal that was intranasally immunized with the formulated extracellular domain of HAPR8; Figure 4 is a graph showing the percentage change in mean body weight of infected mice after infection with influenza virus. = changes in body weight of animals immunized with PEA EDTA-Leu (6) polymer in the formulation buffer (all mice died of viral infection on day 7); 〇 = mice immunized intraperitoneally with virus, positive control Changes in mean body weight of animals administered with intranasal administration of HAPR8-3 and NPPR8 with PEA EDTA-Leu(6)-Zn and poly I:C particles (1 mouse died on day 8 and no production of HA protein) Measured antibody reaction); △ = mice immunized subcutaneously with HAPR8-3 and NPPR8 with PEA EDTA-Leu(6)-Zn and poly I:C particles (except 1 mouse, others are at 8th Figure 5 is the amino acid sequence of the His-tagged nuclear protein from influenza virus strain A/PR/8/34 (Mount Sinai) (SEQ ID NO: 1); Figure 6 is from influenza Amino acid sequence of the HAPR8 extracellular domain antigen of virus strain A/PR/8/34 (Mount Sinai) (SEQ ID NO: 2); Figure 7 is from influenza virus strain A/PR/8/3 4 (Mount The amino acid sequence of the HAPR8-2 His-tagged subfragment antigen of the HA protein of Sinai). The additional underlined portion serves as a signal sequence for bacterial expression and does not appear in the amino acid sequence produced by the bacteria (SEQ ID NO: 3); Figure 8 is from influenza virus strain A/PR/8/34 (Mount) The amino acid sequence of the HAPR83 His-labeled subfragment antigen of the HA protein of Sinai). The additional underlined portion serves as a signal sequence for bacterial expression and does not appear in the amino acid sequence produced by the bacteria 140309.doc-64-201039844 (SEQ ID NO: 4); Figure 9 is from influenza virus strain A/ The amino acid sequence of the His-tagged nuclear protein antigen of VN/1203/2004 (SEQ ID NO: 5);

Figure 10 is the amino acid sequence of the HAVN intracellular domain antigen from influenza virus strain A/VN/i203/2004 (SEQ ID NO: 6); Figure 11 is from influenza virus strain A/VN/1203/ The amino acid sequence of the HAVN-2 His-tagged subfragment of the HA protein of 2004. Additional addition of the line sequence as a signal sequence for bacterial expression and not appearing in the amino acid sequence of the bacteria (SEQ ID NO: 7); and Figure 12 is from influenza virus strain A/VN/1203 The amino acid sequence of the HAVN-3 His-tagged subfragment antigen of the /2004 HA protein. The underlined sequence was added as a signal sequence for bacterial expression and did not appear in the amino acid sequence produced by the bacteria (SEQ ID NO: 8). 〇140309.doc •65· 201039844 Sequence Listing <110> American Mandivis Co., Ltd. <120> Biodegradable Metal Chelating Polymers & Vaccines <130> MEDIV4000-1 <140> 098118871 <141> 2009-06-05 <150>12/437,435 <151> 2009-05-07 <160> 8 <170> Patentln version 3.5

<210> 1 <211> 505 <212> PRT <213> Influenza A virus <400>

Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Asp 15 10 15

Gly Glu Arg Gin Asn Ala Thr Glu lie Arg Ala Ser Val Gly Lys Met 20 25 30 lie Gly Gly He Gly Arg Phe Tyr lie Gin Met Cys Thr Giu Uu Lys 35 40 45

Leu Ser Asp Tyr Glu Gly Arg Leu lie Gin Asn Ser Leu Thr He Glu 50 55 60

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu 65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro lie 85 90 95

Tyr Arg Arg Val Asn Gly Lys Trp Met Arg Glu Leu lie Leu Tyr Asp 100 105 110

Lys Glu Glu lie Arg Arg lie Trp Arg Gin Ala Asn Asn Gly Asp Asp 135 120 125

Ala Thr Ala Gly Leu Thr His Met Met lie Trp His Ser Asn Leu Asn 130 135 140

Asp Ala Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160

Pro Arg Met Cys Ser Leu Met Gin Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu 180 185 190 140309 - Sequence Listing.doc 201039844

Leu Val Arg Met lie Lys Arg Gly lie Asn Asp Arg Asn Phe Trp Arg 195 200 205

Gly Glu Asn Gly Arg Lys Thr Arg He Ala Tyr Glu Arg Met Cys Asn 210 215 220

He Leu Lys Gly Lys Phe Gin Thr Ala Ala Gin Lys Ala Met Met Asp 225 230 235 240

Gin Val Arg Giu Sei Arg Asn Pro Gly Asn Ala Glu Phe Glu Asp Leu 245 250 255

Thr Phe Leu Ala Arg Ser Ala Leu lie Leu Arg Gly Ser Val Ala His 260 265 270

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser GJy 275 280 285

Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly lie Asp Pro Phe 290 295 300

Arg Leu Gin Asn Ser Gin Va] Tyr Ser Leu lie Arg Pro Asn Glu Asn 305 310 315 320

Pro Ala His Lys Ser Gin Leu Val Trp Met Ala Cys His Ser Ala Ala 325 330 335

Phe Glu Asp Leu Arg Val Leu Ser Phe lie Lys Gly Thr Lys Val Leu 340 345 350

Pro Arg Gly Lys Leu Ser Thr Arg Gly Val G]n lie Ala Ser Asn Glu 355 360 365

Asn Met Glu Thr Met Gla Ser Ser Thr Leu Glu Leu Arg Ser Arg Tyr 370 375 380

Trp Ala lie Arg Thr Arg Ser Gly Gly Asn Thr Asn Gin Gin Arg Ala 385 390 395 400

Ser Ala Gly Gin He Ser lie Gin Pro Thr Phe Ser Val Gin Arg Asn 405 410 415

Leu Fro Phe Asp Arg Thr Thr He Met Ala Ala Phe Asn Gly Asn Thr 420 425 430

Glu Gly Arg Thr Ser Asp Met Arg Thr Glu lie lie Arg Met Met Giu 435 440 445

Ser Ala Arg Pro Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe Glu 450 455 460

Leu Ser Asp Glu Lys Ala Ala Ser Pro lie Val Pro Ser Phe Asp Met 465 470 475 480

Ser Asn Glu Gly Ser Tyr Phe Phe Giy Asp Asn A!a Glu G!u Dyr yr Asp 485 490 495 -2- 140309 · Sequence Listing.doc 201039844

Asn Thr Ser His His His His His His 500 505

<210> 2 <211> 514 <2I2> PRT <213> Influenza A virus <400> 2

Met Lys Ala Asn Leu Leu Vai Leu Leu Ser Ala Leu Ala Ala Ala Asp 15 10 15

Ala Asp Thr lie Cys lie Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30

Val Asp Thr Val Leu Glu Lys Asn Va] ThT Va] Thr His Ser Val Asn 35 40 45

Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly lie 50 55 60

Ala Pro Leu Gin Leu Gly Lys Cys Asn lie Ala Gly Trp Leu Leu Gly 65 70 75 80

Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr lie 85 90 95

Val Glu Thr Pro Asn Ser Glu Asn Gly He Cys Tyr Pro Gly Asp Phe 100 105 110 lie Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe 115 120 125

Glu Arg Phe Glu lie Phe Pro Lys Glu Ser Ser Trp Fro Asn His Asn 130 135 140

Thr Asn Gly Vai Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe ^ 145 150 155 160 〇

Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175

Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190

Trp Gly lie His His Pro Pro Asn Ser Lys Glu Gin Gin Asn He Tyr 195 200 205

Gin Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220

Arg Phe Thr Pro Glu lie Ala Glu Arg Pro Lys Val Arg Asp Gin Ala 225 230 235 240

Gly Arg Met Asn Tyr Tyr Trp Thr Leu Uu Lys Pro Gly Asp Thr lie 245 250 255 140309 - Sequence Listing.doc 201039844 lie Phc Glu Ala Asn Gly Asn Leu lie Ala Pro Met Tyr Ala Phe Ala 260 265 270

Leu Ser Arg Gly Phc Gly Ser Gly lie lie Thr Ser Asn Ala Ser Met 275 280 285

His Glu Cys Asn Thr Lys Cys Gin Thr Pro Leu Gly Ala lie Asn Ser 290 295 300

Ser Leu Pro Tyr Gin Asn He His Pro Val Thr lie Gly Glu Cys Pro 305 310 315 320

Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Vai Thr Gly Leu Arg Asn 325 330 335

Thr Pro Ser lie Gin Ser Arg Gly Leu Phe Gly Ala lie Ala Gly Phe 340 345 350

He Glu Gly Gly Ding rp Thr Gly Met lie Asp G]y Ding rp Tyr Gly Ding yr His 355 360 365

His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Lys Ser Thr 370 375 380

Gin Asn Ala lie Asn Gly He Thr Asn Lys Val Asn Thr Val He Glu 385 390 395 400

Lys Met Asn lie Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415

Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430

Asp lie Trp Thr Ding yr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440 445

Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460

Val Lys Ser Gin Leu Lys Asn Asn Ala Lys Glu lie Gly Asn Gly Cys 465 470 475 480

Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 485 490 495

Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu His His His His 500 505 5]0

His His <210> 3 <211> 373 <2I2> PRT <2B> Influenza A virus <400> 3 -4- 140309 - Sequence Listing.doc 201039844

Met Lys Lys Asn He Ala Phe Leu Leu Ala Ser Met Phe Va! Phe Ser 15 10 15 I]e Ala. Thr Asn Ala Tyr Ala Met Lys Ala Asn Leu Leu Val Leu Leu 20 25 30

Ser Ala Leu Ala Ala Ala Asp Ala Asp Thr He Cys lie Gly Tyr His 35 40 45 A)a Asn Asn Ser rihr Asp Thr Val Asp Thr Va! Leu Glu Lys Asn Val 50 55 60

Thr Val Tlir His Ser Va] Asn Leu Leu Glu Asp Ser His Asn Gly Lys 65 70 75 80

Leu Cys Arg Leu Lys Gly lie Ala Pro Leu Gin Leu Gly Lys Cys Asn 85 90 95

He Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro Ϊ00 105 110

Val Arg Ser Trp Ser Tyr He Val Glu Thr Pro Asn Ser Glu Asn Gly 115 120 125 lie Cys Tyr Pro Gly Asp Phe lie Asp Tyr Glu Glu Leu Arg Glu Gin 130 135 140

Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu lie Phe Pro Lys Glu 145 150 155 160

Ser Ser Trp Pro Asn His Asn Thr Asn Gly Val Thr Ala Ala Cys Ser 165 170 175

His Glu Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Glu 180 185 190

Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys 195 200 205

Gly Lys Glu Val Leu Val Leu Trp Gly lie His His Pro Pro Asn Ser 210 215 220

Lys Glu Gin Gin Asn lie Tyr Gin Asn Glu Asn Ala Tyr Val Ser Val 225 230 235 240

Val Ilir Ser Asn Tyr Asn Arg Arg Phe Thr Pro Glu lie Ala Glu Arg 245 250 255

Pro Lys Val Arg Asp Gin Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu 260 265 270

Leu Lys Pro Gly Asp Thr lie lie Phe Glu Ala Asn Gly Asn Leu I1e 275 280 285

Ala Pro Met Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly lie 290 295 300 140309 · Sequence Listing.doc 201039844 lie Thr Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gin Thr 305 310 315 320

Pro Leu Gly Ala lie Asn Ser Ser Leu Pro Ding yr Gin Asn lie His Pro 325 330 335

Val Thr lie Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg 340 345 350

Met Val Thr Gly Leu Arg Asn Thr Pro Ser lie Gin Ser Gly Gly His 355 360 365

His His His His His 370

<210> 4 <211> 254 <212> PRT <2]3>Influenza A virus <400> 4

Met Lys Lys Asn lie Ala Phe Leu Leu Ala Ser Met Phe Val Phe Scr 15 10 15 lie Ala Thr Asn Ala Tyr Ala Lys Gly lie Ala Pro Leu Gin Leu Gly 20 25 30

Lys Cys Asn lie Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Pro 35 40 45

Leu Leu Pro Val Arg Ser Trp Ser Tyr lie Val Glu Thr Pro Asn Ser 50 55 60 G!u Asn Gly lie Cys Tyr Pro Gly Asp Phe lie Asp Dyr Glu Glu Leu 65 70 75 80

Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu lie Phe 85 90 95

Pro Lys Glu Ser Ser Trp Pro Asn His Asn Thr Asn Gly Val Thr Ala 100 105 110

Ala Cys Ser His Glu Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp 115 120 125

Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser Tyr Val 130 135 140

Asn Lys Lys Gly Lys Glu Val Leu Vai Leu Trp Gly lie His His Pro 145 150 155 160

Pro Asn Ser Lys Glu Gin Gin Asn lie Tyr Gin Asn Glu Asn Ala Tyr 165 170 175

Val Ser Val Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr Pro Glu lie 180 185 190 140309 - Sequence Listing.doc 201039844

Ala Glu Arg Fro Lys Val Arg Asp Gin Ala Gly Arg Met Asn Tyr Tyr 195 200 205

Trp Thr Leu Leu Lys Pro Gly Asp Thr lie lie Pbe Glu Ala Asn G3y 210 215 220

Asn Leu lie Ala Pro Met Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly 225 230 235 240

Ser Gly lie lie Thr Ser Ser Gly His His His His His His 245 250

<2]0> 5 <211> 503 <212> PRT <2)3>Influenza A virus <400>5 Ο

Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Gly 15 10 15

Gly Glu Arg Gin Asn Ala Thr Glu He Arg Ala Ser Val Gly Arg Met 20 25 30

Val Ser Gly lie Gly Arg Phe Tyr lie Gin Met Cys Thr Glu Leu Lys 35 40 45

Leu Ser Asp Tyr Glu Gly Arg Leu lie Gin Asn Ser lie T!ir He Glu 50 55 60

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu 65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro lie 85 90 95

Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu lie Leu Tyr Asp 100 105 110

Lys Glu Glu Πε Arg Arg He Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125

Ala Thr Ala Gly Leu Thr His Leu Met lie Trp His Ser Asn Leu Asn 130 135 140

Asp Aia Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160

Pro Arg Met Cys Ser Leu Met Gin Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu 180 185 190

Leu lie Arg Met lie Lys Arg Gly lie Asn Asp Arg Asn Phe Trp Arg 195 200 205 140309 - Sequence Listing.doc 201039844

Gly Glu Asn Gly Arg Arg Thr Arg lie Ala Tyr Glu Arg Met Cys Asn 210 215 220

He Leu Lys Gly Lys Phc Gin ITir Ala Ala Gin Arg Ala Met Met Asp 225 230 235 240

Gin VaJ Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu lie Glu Asp Leu 245 250 255 lie Phe Leu Ala Arg Ser Ala Leu He Leu Arg Gly Ser Val Ala His 260 265 270

Lys Ser Cys Leu Pro Ala Cys Va] Tyr Gly Leu Ala Val Ala Ser Gly 275 280 285

Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly He Asp Pro Phe 290 295 300

Arg Leu Leu Gin Asn Ser CIn Val Phe Ser Leu lie Arg Pro Asn Glu 305 310 315 320

Asn Pro Ala His Lys Ser Gin Leu Val Trp Met Ala Cys His Ser Ala 325 330 335

Ala Phe Glu Asp Leu Arg Val Ser Ser Phe He Arg Gly Thr Arg Val 340 345 350

Val Pro Arg Gly Gin Leu Ser Thr Arg Gly Val Gin lie Ala Ser Asn 355 360 365

Glu Asn McE Glu Ala Met Asp Ser Asn Thr Leu Glu Leu Arg Ser Arg 370 375 3S0

Tyr butyl Ala lie Arg Thr Arg Ser Gly Gly Asn Thr Asn Gin Gin Arg 385 390 395 400

Ala Ser Ala Gly Gin lie Ser Va] G3n Pro Thr Phe Scr Val Gin Arg 405 410 415

Asn Leu Pro Phe Glu Arg Ala Thr lie Met Ala Ala Phe Thr Gly Asn 420 425 430

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu lie lie Arg Met Met 435 440 445

Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe 450 455 460

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro lie Val Pro Ser Phe Asp 465 470 475 480

Met Asn Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu G1u Thr 485 490 495

Ser His His His His His His 140309 - Sequence Listing.doc 500 201039844

<210> 6 <2Π> 517 <212> PRT influenza A virus <400> 6

Met Glu Lys lie Val Leu Leu Phe Ala lie Val Ser Leu Val Lys Ser 15 10 15

Asp Gin fie Cys He Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 20 25 30

Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 35 40 45

Leu G!u Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Vai Lys 50 55 60

Pro Leu He Leu Arg Asp Cys Ser Val Ala Giy Trp Leu Leu Gly Asn 65 70 75 80

Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp Ser Tyr He Val 85 90 95

Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn 100 105 110

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie Asn His Phe Glu 115 120 125

Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser 130 135 140

Leu Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Lys Ser Ser Phe Phe 145 150 155 160

Arg Asn Val Val Trp Leu lie Lys Lys Asn Ser Thr Tyr Pro Thr lie 165 170 175

Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 180 185 190

Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Lys Leu Tyr Gin 195 200 205

Asn Pro Thr Tlir Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 210 215 220

Leu Val Pro Arg lie Ala Thr Arg Ser Lys Val Asn Gly Gin Ser Gly 225 230 235 240

Arg Met Glu Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 245 250 255

Phe Glu Ser Asn Gly Asn Phe lie Ala Pro Glu Tyr Ala Tyr Lys lie 140309 · Sequence Listing.doc 201039844 260 265 270

Val Lys Lys Gly Asp Ser Thr lie Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285

Asn Cys Asn Tlir Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 290 295 300

Met Pro Phe His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys 305 310 315 320

Tyr Val Lys Ser Asn Arg Leu Val Leu Ala ]lir Gly Leu Arg Asn Ser 325 330 335

Pro Gin Arg Glu krg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala lie 340 345 350

Ala Gly Phe lie Glu Gly 〇]y Trp Gin Gly Met Val Asp Gly Trp Tyr 355 360 365

Gly Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380

Glu Ser Thr Gin Lys Ala lie Asp Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 lie lie Asp Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe 405 410 415

Asn Asn Leu Glu Arg Arg lie Glu Asn Leu Asn Lys Lys Met Glu Asp 420 425 430

Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Lea Met 435 440 445

Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu 450 455 460

Tyr Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470 475 480

Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490 495

Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu His 500 505 510

His His His His His 515

<210> 7 <211> 370 <212> PRT <2>3> Influenza A virus <400>

Met Lys Lys Asn lie Ala Phe Leu Leu Ala Ser Met Phe Val Phe Ser •10- 140309- Sequence Listing.doc 201039844 1 5 10 15 lie Ala Thr Asn Ala Tyr Ala Met Glu Lys lie Val Leu Leu Phe Ala 20 25 30 lie Val Ser Leu Vai Lys Ser Asp Gin lie Cys lie Gly Tyr His Ala 35 40 45

Asn Asn Ser Thr Glu Gin Val Asp Thr lie Met Glu Lys Asn Val Thr 50 55 60

Val Thr His Ala Gin Asp lie Leu Glu Lys Lys His Asn Gly Lys Leu 65 70 75 80

Cys Asp Leu Asp Gly Val Lys Pro Leu He Leu Arg Asp Cys Ser Val 85 90 95 Ο

Ala Gly Ding rp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe lie Asn Val 100 105 110

Pro Glu Trp Ser Tyr lie Val Glu Lys Ala Asn Pro Val Asn Asp Leu 115 120 125

Cys Tyr Pro Gly Asp Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu 130 135 140

Ser Arg lie Asn His Phe Glu Lys lie Gin lie lie Pro Lys Ser Ser 145 150 155 160

Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr 165 170 175

Gin Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu He Lys Lys ]80 185 190

Asn Ser Tlu Tyr Pro Thr He Lys Arg Scr Tyr Asn Asn Thr Asn Gin 195 200 205

Glu Asp Leu Leu Val Leu Trp Gly lie His His Pro Asn Asp Ala Ala 210 215 220

Glu Gin Thr Lys Leu Tyr Gin Asn Pro Thr Thr Tyr lie Ser Val Gly 225 230 235 240

Thr Ser Thr Leu Asn Gin Arg Leu Val Pro Arg lie Ala Thr Arg Ser 245 250 255

Lys Val Asn Gly Gin Ser Gly Arg Met Glu Phe Phe Trp Thr lie Leu 260 265 270

Lys Pro Asn Asp Ala lie Asn Phe Glu Ser Asn Gly Asn Phe lie Ala 275 280 285

Pro Glu Tyr Ala Tyr Lys lie Val Lys Lys Gly Asp Ser Thr He Met 290 295 300 • 11 - 140309 - Sequence Listing.doc 201039844

Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gin Thr Pro 305 310 315 320

Met Gly Ala lie Asn Ser Ser Met Pro Phe His Asn lie His Pro Leu 325 330 335

Thr lie Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Ar£ Leu Val Leu 340 345 350

Ala Thr Gly Leu Arg Asn Ser Pro Gin Ser Gly Gly His His His His 355 360 365

His His 370

<210〉 8 <21]> 254 <212> PRT influenza A virus <400> 8

Met Lys Lys Asn He Ala Phe Leu Leu Ala Ser Met Phe Val Phe Ser 15 10 15 lie Ala Thr Asn Ala Tyr Ala Gly Val Lys Pro Leu lie Leu Arg Asp 20 25 30

Cys Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe 35 40 45 lie Asn Val Pro Glu Trp Ser Tyr lie Val Glu Lys Ala Asn Pro Val 50 55 60

Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr Glu Glu Leu Lys 65 70 75 80

His Leu Leu Ser Arg He Asn His Phe Glu Lys lie Gin lie lie Pro 85 90 95

Lys Ser Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser Ala 100 105 110

Cys Pro Tyr Gin Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu 315 120 125 lie Lys Lys Asn Ser Thr Tyr Pro Thr lie Lys Arg Ser Tyr Asn Asn 130 135 140

Thr Asn Gin Glu Asp Leu Leu Val Leu Trp Gly lie His His Pro Asn 145 150 155 160

Asp Ala Ala Glu Gin Thr Lys Leu Tyr Gin Asn Pro Thr Thr Tyr lie 165 170 175

Ser Val Gly Thr Ser Thr Leu Asn Gin Arg Leu Val Pro Arg lie Ala 180 185 290 •12- 140309-Sequence List.doc 201039844

Thr Arg Ser Lys Val Asn Gly Gin Ser Gly Arg Met Glu Phe Phe Trp 195 200 205

Thr lie Leu Lys Pro Asn Asp Ala He Asn Phe Glu Ser Asn Gly Asn 210 215 220

Phe lie Ala Pro Glu Tyr Ala Tyr Lys lie Val Lys Lys Gly Asp Ser 225 230 235 240

Thr lie Met Lys Ser Glu Ser Gly His His His His His His 245 250

O 13- 140309 - Sequence Listing.doc

Claims (1)

201039844 VII. Patent Application Range: 1. A composition comprising at least one of the following polymers or salts thereof: a ruthenium polymer having the formula of formula (I), 9 9 η 9 oh -C-R1- C—NH.9—C-0-R4-. One Q is one death nh R3 point 3 Formula (I) where n is in the range of about 15 to about 150; R1 is -ch2-n(ch2co2h)-r6-n(ch2co2h)-ch2-, wherein R6 is independently selected from the group consisting of: (C2_Ci2) alkyl, _C6H4, (c2-c4) alkoxy a group (c2-c4) alkyl, CH2CH2N(CH2C〇2H)CH2CH2, and a compound having the chemical structure of formula (π) wherein R7 is selected from the group consisting of hydrogen, (Ci-C 2) alkyl and a protecting group, And combinations thereof; R7 R7 R7 —H<p-N—or a π—A—?H-CH2- or a H2C-HC-l5l_CH-CH2-?h2 ch2 ch2 cooh cooh cooh COOH COOH COOH formula (II) The R3 in the n unit is independently selected from the group consisting of hydrogen, (G-C6)alkyl, (C2-C6)alkenyl, (c2-c6)alkynyl, (C6-C10)aryl (CrCd) House, -(CH2)2SCH3, CH2OH, CH(OH) ch3, (ch2)4nh3+, (CH2)3NHC(=NH2+)NH2, 4-imidazolidinium rust (imidazolinium), CH2CO〇-, (CH2 2COO· and combinations thereof; R4 is independently selected from the group consisting of: (C2_C2〇)alkylene, (CrC2〇) 140309.doc 201039844 alkenyl, (c2-c6)alkoxy (c2-c12) Alkyl, ch2ch(oh)ch2, CH2CH(CH2OH), 1, (3), 6-di-dehydrated hexose a bicyclic fragment of an alcohol, a fragment of 1,4-anhydroerythritol, and a combination thereof; CH· _0 H2C 0 a ch 2 %CH Formula (III) or a PEA polymer having the formula of the formula (IV): 9 9 Η Ο ο Η --c-r1-c-nh-cco-r4-occ-nh R3 R3 oo -c-r1-c-nh-ch-r5-nh CO-R2 MO qj where (IV) where n In the range of from about 15 to about 150, m is in the range of from about 0.1 to 0.9; ρ is in the range of from about 0.9 to 0.1; wherein R1 is -CH2-N(CH2C02H)-R6-N(CH2C02H)- CH2-, wherein R6 is independently selected from the group consisting of: (C2_Cl2) alkylene, p_C6H4, (C2-C4) alkoxy (C2-C4) alkyl, CH2CH2N(CH2C02H)CH2CH2, and having the formula (II) Chemical structure wherein R7 is selected from the group consisting of hydrogen, (Cl_Cl2) alkyl, a protecting group, and combinations thereof; R7 R7 HC-N-CH- or -HC--f\|-R7 CH2 ch2 COOH COOH I - - ch-ch2- 41 —h2c—hc—n—ch-ch2-?H2 COOH 1 1 COOH COOH COOH Formula (II) R 2 is independently selected from hydrogen, (Ci_Ci 2 ) alkyl or (C 6 —Ci 〇 ) aryl 140309 .doc -2- 201039844 and a group consisting of protecting groups; the R3 lines in individual n units are independently selected from Groups of the following: nitrogen, (Cl_C6) alkyl, (C2_C6) dilute, (C2-C6) fast radical, (C6-C10) aryl (Ci-CJ alkyl, gas CH2) 2sCH3, CH2〇H, CH(〇H) • CH3, (CH2)4NH3+, (CH2)3NHC(=NH2+)NH2, 4-methylenemi-α, CH 琳 、, CH2C0Cr, (CHACOO. and combinations thereof; R4 is independently free of the following Group consisting of: (CrCy alkylene, (C2_C2〇)alkylene, (C2_C6) alkoxy (C2-C12) alkyl, CH2CH(OH)CH2, CH2CH(CH2〇H) ' structural formula ( a bicyclic fragment of 1,4:3,6-di-dehydrated alcohol, a fragment of 1,4-anhydroerythritol, and a combination thereof; and the R5 is independently selected from the group consisting of (Cl_C4) alkyl group. 2. The composition of claim 1, wherein Ri is _n(CH2C〇2H)_r6_n(CH2C〇2H)-, wherein R6 has the chemical structure of formula (II), wherein R is selected from hydrogen, C^-C! 2) A group consisting of a base and a protecting group. 3. The composition of claim 1 additionally comprising a metal ion that forms a miscellaneous chelating agent with the polymer, the metal ion being selected from the group consisting of Ca2+, Mg2+, Mn2+, C〇2+, Fe2+, Fe3+, Ni2+, Zn2+ And a group of its combination. 4. The composition of claim 2, further comprising at least one cargo molecule selected from the group consisting of: a polar molecule, a His-tagged molecule, a biomolecule, and having Unsaturated regions and/or lone electrons for lipophilic therapeutic molecules comprising a negative microdomain composed of a group of 〇-'S- or N-, and combinations thereof. 5. The composition of claim 4, wherein the at least one load molecule is selected from the group consisting of 140309.doc 201039844 from Paclitaxel, Sirolimus, Everolimus, Dorsey A group of Docetaxel and Biolimus. 6) The composition of claim 4, wherein the at least one load molecule comprises serum albumin. 7. The composition of claim 4, wherein the at least one load molecule comprises a ligand that specifically binds to stem cells, organs or tissues. 8' The composition of claim 4, wherein the at least one load molecule is toxic or specific to the target cell, organ or tissue. 9. The composition of claim 1 additionally comprising a metal forming a complex with the polymer 'the metal is selected from the group consisting of Gd(m) and radioisotopes of Rh, ir, Yt' and wherein the combination The substance is a diagnostic composition. 10. The composition of claim 9, wherein R1 is _n(CH2c〇2H)_r6_n(ch2co2h)-, wherein R6 is CH2CH2N(CH2C〇2H)CH2cH2, and the metal is Gd(III). 11. The composition of claim 7, further comprising at least one load molecule that kills or targets the cell, selected from the group consisting of a polar molecule, a biomolecule, a His-tagged molecule, and having an unsaturated The region and/or the lone electron is a lipophilic molecule of a negative polarity composed of a group containing 〇_, S^N_. 12. A method of preparing nanoparticle, the method comprising: a) contacting the following together in an aqueous solution under polycondensation conditions: 1) at least one polymer of claim 1; 2) selecting Ca2+, Mg2+, Mn2+ , C〇2+, Fe2+&Fe3+, 140309.doc 201039844 Zn, Nl and Gd3+ groups of metal ions; and 3) aprotic polar solvent; b) formation of the polymer in the solution Nanoparticles of the non-covalent complex of the metal cation; and _C) the nanoparticles are obtained by separating from the solution by size exclusion. 13. The method of claim 12, wherein the solution further comprises at least one load molecule selected from the group consisting of: a polar molecule, a biomolecule, a His-tagged molecule, and having an unsaturated region and/or a lone electron The lipophilic molecule having a negative microdomain composed of a group of ruthenium- and S-, and wherein the complex formed in the nanoparticle at S-hai or the like additionally comprises the at least one supported molecule. 14. The method of claim 12, wherein the solution further comprises the amino acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. The method of claim 12, wherein the His-tagged molecule comprises an amino acid sequence comprising a 病 pathogenic epitope. 16. The method of claim 15, wherein the His-tagged molecule is recombined in the solution. 17. The method of claim 15, wherein the His-tagged molecule is recombined in the bacterium. 18. A composition comprising: a) a bioactive agent selected from the group consisting of polyethylene glycol or polyethylene glycol, polysaccharides, lipids, biomacromolecules, and water-insoluble drugs; and b) Polymer of claim 1, 140309.doc 201039844 # t The polymer is flanked on both sides of the composition of which is a linear polymer of the bioactive agent. 19. 20. The bioactive agent is a polymeric immunosuppressant as claimed in claim 18. The composition of claim 19, further comprising: c) a metal ion selected from the group consisting of Ca2+, Mg2+, Mn2+, Co2+, Fe2+ & Fe3+, Zn2+, Ni; and the metal ion is maintained at d) An amino acid sequence comprising a pathogenic epitope, wherein the metal ion and the amino acid sequence are linked to the polymer via a non-covalent complex formed with the polymer. 140309.doc