CN101039620A - Dendrimer based compositions and methods of using the same - Google Patents

Abstract

The present invention relates to novel therapeutic and diagnostic dendrimers. In particular, the present invention is directed to dendrimer based multifunctional compositions and systems for use in disease diagnosis and therapy (e.g., cancer diagnosis and therapy). The compositions and systems comprise one or more components for targeting, imaging, sensing, and/or providing a therapeutic or diagnostic material and monitoring the response to therapy of a cell or tissue (e.g., a tumor).

Description

Compoistion and method of use based on dendritic macromole

The present invention requires the U.S. Provisional Patent Application serial number US60/604 of submission on August 25th, 2004, the U.S. Provisional Patent Application serial number US60/690 that on June 15th, 321 and 2005 submitted to, 652 priority intactly is incorporated herein by reference these documents.

The present invention partly is subjected to the subsidy of NIH Contract NO1-CO-97111 and NCI ContractNOl-CM-97065-32.Government can have some right among the present invention.

Invention field

The present invention relates to new treatment and diagnosis dendritic macromole (dendrimers).Especially, the present invention relates to the multifunctional compositions that is used for medical diagnosis on disease and therapy and system's (for example cancer diagnosis and therapy) based on dendritic macromole.These compositionss and system comprise one or more be used for targeting, imaging, sensing and/or treatment is provided or diagnostic substances and monitoring cell or tissue (for example tumor) to the component of the reaction of therapy.

Background of invention

Cancer is deputy major causes of death in the U.S., and making just has 1 cancer in per 4 deaths.In 1997, the diagnosis sum of according to estimates new pulmonary carcinoma, breast carcinoma, carcinoma of prostate, colorectal carcinoma and ovarian cancer was about 200 ten thousand.Owing to increasing all the time, reasonably estimate it is the sickness rate meeting sustainable growth of cancer U.S. aging crowd.

Make at present and treat cancer in various manners, comprise operation, radiation and chemotherapy.Type, position and the distribution of cancer depended in the selection of therapeutic modality.For example, many common tumors are difficult to react to available therapy such as colon cancer.

With regard to the tumor type that present method is reacted, only the part cancer is to the sufficient reacting of therapy.In addition, although the therapy of many cancers is improved, there is serious adverse in the activating agent of up-to-date use.These side effect have limited the application of chemotherapeutics usually and have caused most of cancer to be selected without any treatment.The treatment of other type is attempted, and can turn out to be such as gene therapy or immunotherapy to have more specific and than the few side effects of chemotherapy.Yet although shown certain progress in several clinical trials, the practical application of these means is still limited at present.

Although it is the success of existing therapy is limited, developed to the understanding of the fundamental biological knowledge of tumor cell.Identify the cell growth that relates to sexual activity of tumor cell transformed and change at present and write down a plurality of steps in the carcinogenesis of several human tumors (for example, referring to Isaacs, Cancer 70:1810 (1992)).Identified the oncogene of the cell growth that causes to regulate and it has been characterized by genetic origin and function.Characterized in detail and regulated the specificity approach of cell replication cycle and cloned and characterized the protein that relates in this adjusting.In addition, sets forth in detail mediating apoptosis oppositely regulate the molecule (Kerr etc., Cancer 73:2013 (1994)) of cell growth.Confirmed at present to handle these cells regulate approach can stop growth in the tumor cell and induce wherein apoptosis (for example, referring to Cohen and Tohoku, Exp.Med., 168:351 (1992); With J.Natl.Cancer Inst. such as Fujiwara, 86:458 (1994)).Cell in the control tumor cell is grown and duplicated is important treatment target.

Although obtained compelling achievement, before these therapies can be applied to the interior therapeutic cancerous cell, still there are many obstructions.For example, these therapies need identify that the specificity pathophysiology of individual specific tumors cell changes.This just requires the mechanicalness infringement (biopsy) of tumor and generally diagnoses by cell in vitro cultivation and test.Before can selecting and carrying out therapy, must analyze tumor phenotypes then.This class step is consuming time, complicated and expensive.

To comparing with normal cell, having optionally to tumor cell, there is demand in Therapeutic Method.Present therapy only has relative specificity to the treatment cell.Although cancer target has solved this selective problems, it remains insufficient, because tumor does not have unique antigen.In addition, it is desirable to therapy and should have parallel working preventing to select several different mechanism of action of resistance tumor, and after checking knub position and type, can remove by the clinicist.At last, it is desirable to, therapy should be able to make the clinicist identify the disease of residual or minimum level at once before and after treatment, and monitors the reaction to therapy.This is crucial, because several residual cell just may cause regrowth, or worsens, and produces the tumor that therapy is produced resistance.Identify that disease residual when therapy finishes (the long back of non-tumor regrowth) can help eradicating a little remaining tumor cell.

Therefore, ideal therapy should have target tumor, makes degree (the being neoplasm metastasis) imaging of tumor and identifies the ability that has therapeutic agent in tumor cell.Therefore, need make the clinicist can select to treat the therapy of molecule unusually,, write down reaction, and identify residual disease therapy so that in abnormal cell, activating therapeutic agent based on the pathophysiology in the tumor cell.

Summary of the invention

The present invention relates to new treatment and diagnosis dendritic macromole.Especially, the present invention relates to the multifunctional compositions and the system that are used for medical diagnosis on disease and therapy (for example cancer diagnosis and therapy) based on dendritic macromole.These compositionss and system comprise one or more targeting, imaging, sensing and/or treatment is provided or diagnostic substances and monitoring cell or tissue (for example tumor) to the component of the reaction of therapy.

Therefore; in certain embodiments; the invention provides the compositions that comprises dendritic macromole; described dendritic macromole comprises the 5th generation 5 (G5) dendritic macromole (for example polyamidoamine (polyamideamine) (PAMAM), poly-propylamine (POPAM) or PAMAM-POPAM dendritic macromole) of partial acetylation, and this dendritic macromole further comprises one or more functional groups.The present invention is not limited to the application of G5 dendritic macromole.In certain embodiments, one or more functional groups comprise therapeutic agent, targeting agent and/or preparation.In certain embodiments, at least a in the functional group puted together by ester bond and dendritic macromole.In preferred embodiments, therapeutic agent comprises chemotherapy compound (for example methotrexate).In certain preferred aspects, chemotherapy compound is puted together by ester bond and dendritic macromole.In certain preferred aspects, the targeting agent comprises folic acid.In other embodiment preferred, preparation comprises Fluorescein isothiocyanate or other detectable label.In certain embodiments, functional group is one of therapeutic agent, targeting agent or biological monitoring agent.In certain embodiments, G5 dendritic macromole and functional group put together.In certain embodiments, put together and comprise covalent bond, ionic bond, metallic bond, hydrogen bond, van der Waals' bond, ester bond or amido link.

In certain embodiments of the invention, therapeutic agent includes but not limited to chemotherapeutics, resist-causes the tumor agent, anti-angiogenicization agent, tumor inhibitor, antimicrobial or comprise the proteic expression of nucleic acids construct of coding treatment, although the present invention is not limited to the character of therapeutic agent.In other embodiments, be selected from protecting group to photo-labile, to radiating unsettled protecting group and to the protecting group protection therapeutic agent of the unsettled protecting group of enzyme.In certain embodiments, chemotherapeutics is selected from platinum complexes, verapamil, podophyllotoxin, carboplatin, procarbazine, mechloroethamine, cyclophosphamide, camptothecine, ifosfamide, melphalan, chlorambucil, busulfan (bisulfan), nitroso ureas (nitrosurea), amycin, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin (plicomycin), mitomycin, bleomycin, etoposide, tamoxifen, paclitaxel, taxol, transplatinum, 5-fluorouracil, vincristin, the group that vinblastine and methotrexate are formed, but be not limited to them.In certain embodiments, resist-cause the tumor agent and comprise antisensenucleic acids (for example RNA molecule).In certain embodiments, antisensenucleic acids comprises the complementary sequence with the RNA of oncogene.In preferred embodiments, oncogene includes but not limited to: abl, Bcl-2, Bcl-xL, erb, fms, gsp, hst, jun, myc, neu, raf; Ras, ret, src or trk.In certain embodiments, the proteic nucleic acid coding factor of coding treatment includes but not limited to tumor suppressor protein, cytokine, receptor, apoptosis inducers and differentiation agent.In preferred embodiments, tumor suppressor protein includes but not limited to BRCA1, BRCA2, C-CAM, p16, p21, p53, p73, Rb and p27.In preferred embodiments, cytokine includes but not limited to GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, beta-interferon, gamma interferon and TNF.In preferred embodiments, receptor includes but not limited to CFTR, EGFR, estrogen receptor, IL-2 receptor and VEGFR.In preferred embodiments, apoptosis inducers includes but not limited to AdE1B, Bad, Bak, Bax, Bid, Bik, Bim, Harakid and ICE-CED3 protease.In certain embodiments, therapeutic agent comprises short-decayed radiosiotope.

The present invention is not limited to the type (for example puting together with dendritic macromole of the present invention) of used anticarcinogen or chemotherapeutics.In fact, expection can be used for various anticarcinogen of the present invention and chemotherapeutics includes but not limited to: acivicin; Aclarubicin; The hydrochloric acid acodazole; Acronine; Adozelesin; Doxorubicin; Aldesleukin; Alitretinoin; Allopurinol sodium; Altretamine; Ambomycin; The acetic acid ametantrone; Aminoglutethimide; Amsacrine; Anastrozole; AnnonaceousAcetogenins; Anthramycin; Asimicin; Asparaginase; Asperlin; Azacitidine; Azatepa; Azotomycin; Batimastat; Benzodepa; Bexarotene; Bicalutamide; Bisantrene hydrochloride; Two methanesulfonic acid bisnafides; Bizelesin; Bleomycin Sulphate; Brequinar sodium; Bropirimine; Bullatacin; Busulfan; Cabergoline; Actinomycin C; Calusterone; Caracemide; Caracemide; Carboplatin; Carmustine; Carubicin hydrochloride; Carzelesin; Cedefingol; Celecoxib; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; The methanesulfonic acid crisnatol; Cyclophosphamide; Cytosine arabinoside; Dacarbazine; DACA (N-[2-(dimethyl-amino) ethyl] acridine-4-Methanamide); Actinomycin D; Daunorubicin hydrochloride; Daunorubicin; Decitabine; Denileukin diftitox; Dexormaplatin; Dezaguanine; The methanesulfonic acid Dezaguanine; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin hydrochloride; Droloxifene; Droloxifene citrate; Dromostanolone propionate; Duazomycin; Edatrexate; Eflornithine hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin hydrochloride; Erbulozole; Esorubicin hydrochloride; Estramustine; Estramustine phosphate sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; The phosphoric acid etoposide; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; The fluorouracil deoxynucleoside; Fludarabine phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin sodium; FK-317; FK-973; FR-66979; FR-900482; Gemcitabine; Gemcitabine hydrochloride (GeimcitabineHydrochloride); Gemtuzumab Ozogamicin (Gemtuzumab Ozogamicin); Gold Au 198; Goserelin acetate; Guanacone; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Ilmofosine; Intederon Alpha-2a; Interferon Alpha-2b; Interferon alfa-n1; Alferon N; Interferon beta-Ia; Interferon gamma-Ib; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprorelin acetate; Liarozole hydrochloride; Lometrexol sodium; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytansine; Mustine hydrochlcride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Methoxsalen; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitochromine mitocromine B-35251; Mitogillin; Mitomalcin; Mitomycin; Ametycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate Disodium; Pegaspargase; Peliomycin; Neostigmine bromide; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; The hydrochloric acid piroxantrone; Plicamycin; Plomestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofurin; Riboprine; Mabthera; Rogletimide; Rolliniastatin; Safingol; The hydrochloric acid Safingol; Samarium/Lexidronam; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Squamocin; Squamotacin; Rufocromomycin; Streptozocin; Strontium chloride Sr 89; Sulofenur; Talisomycin; Taxane (Taxane); Taxane (Taxoid); Tecogalan sodium; Ftorafur; Teloxandrone hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; ITG; Thioguanine; Plug is for group; Thymitaq; Tiazofurine; Tirapazamine; Thunder is for bent thiophene; TOP-53; Topotecan hydrochloride; Toremifene Citrate; Trastuzumab; Trestolone acetate; The phosphoric acid triciribine; Trimetrexate; The glucuronic acid trimetrexate; Triptorelin; Tubulozole hydrochloride; Uracil mustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine; Vinblastine sulfate; Vincristine; Vincristine sulfate; Vindesine; Vindesine sulfate; The sulphuric acid vinepidine; The sulphuric acid vinglycinate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrosidine sulfate; The sulphuric acid vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2 '-deoxyformycin; 9-aminocamptothecin; Raltitrexed; N-propargyl-5, the 8-two denitrogenations folic acid (dideazafolic ac d) of mixing; 2-chloro-2 '-arabinose (arabino)-fluoro-2 '-deoxyadenosine; 2-chloro-2 '-deoxyadenosine; Anisomycin; Trichostatin A; HPRL-G129R; CEP-751; Linomide; Mustard gas; Chlormethine (nitrogen mustard) (chlormethine (mechlorethamine)); Cyclophosphamide; Melphalan; Chlorambucil; Ifosfamide; Busulfan; N-methyl-N-nitrosourea (MNU); N, N '-two (2-chloroethyl)-N-nitroso ureas (BCNU); N-(2-chloroethyl)-N '-cyclohexyl-N-nitroso ureas (CCNU); N-(2-chloroethyl)-N '-(trans-the 4-methylcyclohexyl-N-nitroso ureas (MeCCNU); N-(2-chloroethyl)-N '-(diethyl) ethylphosphonic acid-N-nitroso ureas (fotemustine); Streptozotocin; Dacarbazine (diacarbazine) (DTIC); Mitozolomide; The temozolomide; Plug is for group; Ametycin; AZQ; Adozelesin; Cisplatin; Carboplatin; Ormaplatin; Oxaliplatin; C1-973; DWA 2114R; JM216; JM335; Two (platinum); Tomudex; Azacitidine; Cytosine arabinoside; Gemcitabine; The 6-mercaptopurine; The 6-thioguanine; Hypoxanthine; Teniposide; 9-aminocamptothecin; Hycamtin; CPT-11; Doxorubicin; Daunorubicin; Epirubicin; Darubicin; Mitoxantrone; Losoxantrone; Actinomycin D (Dactinomycin) (actinomycin D (Actinomycin D)); Amsacrine; Pyrazoloacridine; Alltrans retinol; 14-hydroxyl-retroretinol; All-trans-retinoic acid; N-(4-hydroxyphenyl) retinamide; 13-cis tretinoin; 3-methyl TTNEB; 9-cis tretinoin; Fludarabine (2-F-ara-AMP); And 2-chlorodeoxyadenosine (2-Cda).

Other anticarcinogen and chemotherapeutics comprise that antiproliferative agents (for example different thiosulfuric acid piritrexim), anti-prostate hyperplasia medicine (for example sitogluside), benign prostate increase therapeutic agent (for example tamsulosin hydrochloride), prostate growth inhibitor (for example pentomone) and radioreagent.

Other anticarcinogen and chemotherapeutics can comprise anticancer additional hardening agent, comprise tricyclics (for example imipramine, desipramine, amitriptyline, clomipramine, trimeprimine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); Non--tricyclics (for example Sertraline, trazodone and citalopram); Ca ⁺⁺Antagonist (for example verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (for example prenylamine, trifluoperazine and clomipramine); Amphotericin B; Triparanol analog (for example tamoxifen); Anti-arrhythmic (for example quinidine); Antihypertensive (for example reserpine); Thiol depleters (for example buthionine and sulfoximine) and multiple drug resistance alleviant are such as CremaphorEL.

Other anticarcinogen and chemotherapeutics are selected from the anticarcinogen and the chemotherapeutics of the group of following composition for those: annonaceous acetogenins; Asimicin; Rolliniastatin; Guanacone, squamocin, bullatacin; Squamotacin; Taxanes; Paclitaxel; Gemcitabine; Methotrexate FR-900482; FK-973; FR-66979; FK-317; 5-FU; FUDR; FdUMP; Hydroxyurea; Docetaxel; Discodermolide; Epothilones; Vincristine; Vinblastine; Vinorelbine; Meta-pac; Irinotecan; SN-38; 10-OHcampto; Hycamtin; Etoposide; Doxorubicin; Flavopiridol; Cis-Pt; Carbo-Pt; Bleomycin; Ametycin; Plicamycin; Capecitabine; Cytosine arabinoside; 2-C1-2 ' deoxyadenosine; Fludarabine-PO.sub.4; Mitoxantrone; Mitozolomide; Pentostatin; And Raltitrexed.

Other anticarcinogen and chemotherapeutics comprise taxanes (for example paclitaxel and docetaxel).In certain embodiments, anticarcinogen and chemotherapeutics comprise tamoxifen or aromatase inhibitor Arimidex (for example Anastrozole).

In certain embodiments of the invention, the biological monitoring agent comprises the reagent (for example directly or indirectly measuring the reaction of cytokine or therapeutic-induced) of measuring the therapeutic agent effect, yet the present invention is not limited to the character of biological monitoring agent.In certain embodiments, described monitoring agent can be measured the amount of therapeutic agent or detect the apoptosis that is caused by therapeutic agent.

In certain embodiments of the invention, preparation comprises radioactive label, includes but not limited to ¹⁴C, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ¹²⁵I, ¹³¹I, ¹¹¹Ln, ¹⁵²Eu, ⁵⁹Fe, ⁶⁷Ga, ³²P, ¹⁸⁶Re, ³⁵S, ⁷⁵Se, Tc-99m and ¹⁷⁵Yb.In certain embodiments, preparation comprises the fluorescence body.In a preferred embodiment, preparation is Fluorescein isothiocyanate or 6-TAMARA.

In certain embodiments of the invention, the targeting agent includes but not limited to antibody, receptors ligand, hormone, vitamin and antigen, and but, the present invention is not limited to the character of targeting agent.In certain embodiments, antibody has specificity to disease specific antigen.In certain preferred aspects, disease specific antigen comprises tumor specific antigen.In certain embodiments, receptors ligand includes but not limited to the part of CFTR, FGFR, estrogen receptor, FGR2, folacin receptor, IL-2 receptor, glycoprotein and VEGFR.In a preferred embodiment, receptors ligand is a folic acid.Can be used for other embodiment of the present invention and be described in U.S. Pat 6,471,968 and WO 01/87348 in, these documents intactly are incorporated herein by reference separately.

In certain embodiments, dendritic macromole of the present invention (for example G5PAMAM dendritic macromole) contains lip-deep 2-250, a 10-200 or 100-150 response location (for example referring to embodiment 13).In preferred embodiments, response location comprises primary amine groups.In certain embodiments, dendritic macromole contains 50-250 response location.In certain embodiments, dendritic macromole comprises 150-400 response location.In preferred embodiments, response location and functional group put together, and described functional group includes but not limited to therapeutic agent (for example methotrexate), targeting agent (for example folic acid), preparation (for example FITC) and biological monitoring agent.

In certain embodiments, on single dendritic macromole, can form any one (for example therapeutic agent) in the functional group of a plurality of copies.Therefore, in certain embodiments, single dendritic macromole comprises the single functional group (for example therapeutic agent, such as methotrexate) of 2-100 copy.In certain embodiments, dendritic macromole comprises 2-5,5-10, the single functional group of 10-20 or 20-50 copy.In certain embodiments, dendritic macromole comprises 5-20 copy.In certain embodiments, dendritic macromole comprises functional group's (for example therapeutic agent, targeting agent or preparation) of 50-100 or 100-200 copy.In certain embodiments, dendritic macromole comprises the functional group of copy more than 200.The present invention further provides the dendritic macromole of a plurality of copies that comprise two or more different functional groups.In certain embodiments, the invention provides the functional group that comprises one type (therapeutic agent for example, such as methotrexate, or in other targeting agent discussed in this article any one) a plurality of copies (2-10 for example, 5-10,10-15,15-50,50-100,100-200 or copy more than 200) and second in a plurality of copies (2-10 for example, the 15-50 of functional group's (for example targeting agent is such as in folic acid or other targeting agent as herein described any one) of type, 50-100,100-200 or more than 200 the copy) dendritic macromole.In certain embodiments, dendritic macromole comprises a plurality of copies of 2-10 kind different functional groups.For example, in certain embodiments, (for example represent that dendritic macromole can contain 100-150 diverse location (response location for example based on the data that produce in the R﹠D process of the present invention, such as primary amine groups), the data of puting together functional group (for example referring to embodiment 13), dendritic macromole can contain the therapeutic agent (for example methotrexate) of 2-100 copy, the preparation (for example FITC or 6-TAMARA) of the targeting agent (for example folic acid) of 2-100 copy and 2-100 copy.

The present invention also provides the method for preparing dendritic macromole, and this method comprises one or more steps (with random order): acetylation G5 dendritic macromole and the acetylizad dendritic macromole of generating portion; The dendritic macromole of preparation (for example Fluorescein isothiocyanate) and partial acetylation is puted together and generated the simple function dendritic macromole; The targeting agent (for example folic acid) and the simple function dendritic macromole of partial acetylation are puted together so that generate the difunctionality dendritic macromole; The difunctionality dendritic macromole of (+)-2,3-Epoxy-1-propanol and partial acetylation is puted together; And the therapeutic agent (for example methotrexate) and difunctionality ization (glycidylated) the difunctionality dendritic macromole of partial acetylation are puted together.In preferred embodiments, produce the G5 dendritic macromole according to the following step: (a) obtain initiator core aliphatic diamine; (b) carry out reacting with the Michael of Michael acceptor; (c) with single diamidogen NH2-(CH2) that protects _n-NHPG (n=1-10) condensation; (d) repeating step (a)-(c); Wherein in the amide forming process in per generation, use the diamidogen of single protection.The character of the initiator core aliphatic diamine that the present invention is not limited to select.In certain embodiments, initiator core aliphatic diamine is selected from the group that comprises following material, but is not limited to them: NH2-(CH2) _n-NH2 (n=1-10); NH2-(CH2) _n-NHPG, (n=1-10); NH2-((CH2) _nNH2) ₃(n=1-10); Be not substituted or replace 1,2-; 1,3-; Or 1,4-phenylene two-just-alkylamine.In a preferred embodiment, the Michael acceptor is an acrylic acid methyl ester..In certain embodiments; used protecting group (PG) is selected from the group that comprises following material, but is not limited to them: tert-butoxy carbamate (N-t-Boc), allyloxy carbamate (N-Alloc), benzylamino formic acid esters (N-Cbz), 9-fluorenyl methyl carbamate (FMOC) or phthalimide (Phth).

The present invention also provides the compositions that comprises dendritic macromole, and described dendritic macromole comprises protected core diamidogen.In certain embodiments, dendritic macromole comprises polyamidoamine (PAMAM), poly-propylamine (POPAM) or PAMAM-POPAM dendritic macromole.In particularly preferred embodiments, the core diamidogen is single protection.In certain embodiments, the core diamidogen is NH2-(CH2) _n-NHPG (n=1-10).In preferred embodiments, protected core diamidogen is NH2-CH2-CH2-NHPG.In certain embodiments; protected core diamidogen comprises protecting group (PG); protecting group is selected from and comprises following group, but is not limited to them: tert-butoxy carbamate (N-t-Boc), allyloxy carbamate (N-Alloc), benzylamino formic acid esters (N-Cbz), 9-fluorenyl methyl carbamate (FMOC) or phthalimide (Phth).In preferred embodiments, dendritic macromole is a partial acetylation.In particularly preferred embodiments, dendritic macromole and functional group put together.

The present invention also provides preparation to comprise the method for the dendritic macromole of protected core diamidogen, and this method comprises the following step: the initiator core aliphatic diamine NH2-(CH2) that a) uses single protection _n-NHPG, (n=1-10); B) carry out reacting with the Michael of Michael acceptor; (c) with the singly equivalent condensation of the diamidogen of protection; (d) repeating step (a)-(c); Wherein in the amide forming process in per generation, use the initiator core aliphatic diamine of single protection.In a preferred embodiment, the Michael receptor is an acrylic acid methyl ester..In certain embodiments, repeat to produce the covalently bound radial shell of the repetitive that has surface amino groups of a new generation (G=1-10) at every turn.In certain embodiments, protecting group (PG) comprises tert-butoxy carbamate (N-t-Boc), allyloxy carbamate (N-Alloc), benzylamino formic acid esters (N-Cbz), 9-fluorenyl methyl carbamate (FMOC) or phthalimide (Phth).

The present invention also provides preparation to comprise the method for compositions of dendritic macromole, and this method comprises the following step: the initiator core aliphatic diamine that a) uses single protection; B) carry out reacting with the Michael of Michael acceptor; (c) with single diamidogen NH2-(CH2) that protects _n-NHPG, (n=1-10) condensation; (d) repeating step (a)-(c); Wherein in the amide forming process in per generation, use the diamidogen of single protection.The character of the initiator core diamidogen that the present invention is not limited to select.In certain embodiments, initiator core aliphatic diamine is selected from the group that comprises following material, but is not limited to them: NH2-(CH2) _n-NH2 (n=1-10), NH2-((CH2) _nNH2) ₃(n=1-10); Be not substituted or replace 1,2-; 1,3-; Or 1,4-phenylene two-just-alkylamine.In a preferred embodiment, the Michael acceptor is an acrylic acid methyl ester..In certain embodiments, repeat to produce the covalently bound radial shell of the repetitive that has surface amino groups of a new generation (G=1-10) at every turn.In certain embodiments, protecting group (PG) comprises tert-butoxy carbamate (N-t-Boc), allyloxy carbamate (N-Alloc), benzylamino formic acid esters (N-Cbz), 9-fluorenyl methyl carbamate (FMOC) or phthalimide (Phth).

The present invention also provides the method for treatment disease (for example cancer or infectious disease), comprises to suffer from or the experimenter of susceptibility to disease treats the compositions that comprises dendritic macromole of the present invention of effective dose.In preferred embodiments, dispose dendritic macromole of the present invention, make them be easy to from the experimenter, remove (for example so that few to detecting toxicity hardly under effective dose).In certain embodiments, described disease is a neoplastic disease, and it is selected from but is not limited to leukemia, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin, the non-hodgkin's disease, multiple myeloma, idiopathic macroglobulinemia disease, heavy chain disease, solid tumor, sarcoma and cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterus carcinoma, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, durosarcoma, melanoma and neuroblastoma retinal neuroblastoma (neuroblastomaretinoblastoma).In certain embodiments, described disease is an inflammatory diseases, it is selected from the group that following disease is formed, but is not limited to them: eczema, inflammatory bowel, rheumatoid arthritis, asthma, psoriasis, ischemia/reperfusion injury, ulcerative colitis and adult respiratory distress syndrome.In certain embodiments, described disease is a viral disease, and it is selected from the group that following disease is formed, but is not limited to them: the viral disease that is caused by influenza virus down: hepatitis B virus; Hepatitis C virus; Rotavirus; Human immunodeficiency virus I type (HIV-I); Human immunodeficiency virus II type (HIV-II); Human T lymphotrophic virus I type (HTLV-I); Human T lymphotrophic virus II type (HTLV-II); AIDS; DNA viruses is such as hepatitis B virus and hepatitis C virus; Parvovirus is such as adeno associated virus and cytomegalovirus; Papovavirus is such as human papillomavirus, polyoma virus and SV40; Adenovirus; Herpesvirus is such as herpes simplex I type (HSV-I), herpes simplex II type (HSV-II) and Epstein-Barr virus; Poxvirus is such as variola (variola) and vaccinia virus; And RNA viruses, such as human immunodeficiency virus I type (HIV-I), human immunodeficiency virus II type (HIV-II), human T lymphotrophic virus I type (HTLV-I), human T lymphotrophic virus II type (HTLV-II), influenza virus, Measles virus, rabies virus, celestial platform (Sendai) virus, picornavirus, such as picorna virus, Coxsackie virus, rhinovirus, reovirus, Togaviruses, such as german measles virus (German measles) and Semliki Forest virus, arbovirus and hepatitis A virus.

The present invention also provides the method for treatment disease; comprise and suffer from or the experimenter of susceptibility to disease treats the compositions that comprises dendritic macromole of the present invention of effective dose; described dendritic macromole comprises G5PAMAM, POPAM or the PAMAM-POPAM dendritic macromole of partial acetylation; this dendritic macromole further comprises one or more functional groups, and described one or more functional groups are selected from the group that therapeutic agent, targeting agent and preparation are formed.In certain embodiments, described disease is a neoplastic disease.In certain embodiments, described neoplastic disease is selected from leukemia, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin, the non-hodgkin's disease, multiple myeloma, idiopathic macroglobulinemia disease, heavy chain disease, solid tumor, sarcoma and cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterus carcinoma, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, durosarcoma, the group that melanoma and neuroblastoma retinal neuroblastoma are formed.

The present invention also provides the method that changes experimenter's tumor growth, comprising provides the compositions that comprises dendritic macromole, described dendritic macromole comprises the dendritic macromole of partial acetylation, this dendritic macromole further comprises one or more functional groups, and described one or more functional groups are selected from the group that therapeutic agent, targeting agent and preparation are formed; And under the condition that makes tumor growth obtain changing, give the experimenter described compositions.In certain embodiments, change comprises and suppresses experimenter's tumor growth.In certain embodiments, change comprises the tumor size that reduces among the experimenter.Compositions and the chemotherapeutics or the anticarcinogen co-administered that will comprise in certain embodiments, dendritic macromole.In certain embodiments, changing tumor growth makes tumor to chemotherapy or resist-cause the oncotherapy sensitivity.

Accompanying drawing is described

Accompanying drawing 1 has described to be used for (A) traditional method and (B) comparison of the method for certain embodiments of the invention of synthetic PAPAM dendritic macromole.

Accompanying drawing 2 has been described the preferred protecting group (PG) in protected core texture territory.

It is phenylenediamine N-((CH2) that accompanying drawing 3 has been described to work as core two cases _n-NH2) ₃(n=1-10) the core diamidogen the time.

Accompanying drawing 4 has been described the phenylenediamine of accompanying drawing 3, but have substituent group, wherein R and R1 are independently selected from hydrogen, C1-C6 straight or branched alkyl, C3-C6 cycloalkyl, are not substituted or by C1-C6 alkyl, C1-C6 alkoxyl, 1, the C5-C10 aryl that 3-dioxolanyl, tri haloalkyl, carboxyl, C1-C6 dialkylamino, C1-C6 sulfanatoalkyl, C1-C6 sulfonamides alkyl or C1-C6phosphanatoalkyl replace.

Accompanying drawing 5 has been described by the catalytic reduction that is purchased the two acetonitriles of phenylene is synthesized phenylenediamine.

Accompanying drawing 6 has described to be used to generate the synthetic schemes of multifunctional G5PAMAM dendritic macromole.

Accompanying drawing 7 has been described the potentiometric titration curve of G5PAMAM dendritic macromole.

Accompanying drawing 8 has been described the gel permeation chromatography elution profile (eluograms) of partial acetylation carrier and end-product, wherein 90 ° eclipsed be RI signal and laser light scattering signal.

Accompanying drawing 9 has been described the theory of G5PAMAM dendritic macromole and damaged chemical constitution.

Accompanying drawing 10 has been described (A) H1-NMR spectrum of G5-Ac2 dendritic macromole and (B) HPLC elution profile.

Accompanying drawing 11 has been described the chemical constitution of Fluorescein isothiocyanate, folic acid and methotrexate, and the group that wherein is used to put together is by the asterisk labelling.

Accompanying drawing 12 has been described the proton N MR imaging of Fluorescein isothiocyanate, folic acid and methotrexate.

Accompanying drawing 13 has been described the G5-Ac at 305nm place (A) ²-FITC-OH-MTX ^e(B) G5-Ac ³-FITC-OH-MTX ^eThe HPLC elution profile.

Accompanying drawing 14 has been described G5-Ac ²-FITC-FA-OH-MTX ^eH1-NMR spectrum.

Accompanying drawing 15 has been described the G5-Ac-FITC-FA-OH-MTX at the 305nm place ^eThe HPLC elution profile.

Accompanying drawing 16 has been described the UV spectrum of free Fluorescein isothiocyanate, folic acid and methotrexate.

Accompanying drawing 17 has been described G5-Ac, G5-Ac ³-FITC, G5-Ac ³-FITC-FA and G5-Ac ³The UV spectrum of-FITC-FA-MTXe.

Accompanying drawing 18 described G5-FITC-FA-MTX dose dependent in the KB cell bonded (A) thick and (B) standardization fluorescence.

Accompanying drawing 19 has been described free FA effect to the picked-up of G5-FITC-FA and G5-FITC-FA-MTX in the KB cell of expressing high and low FA receptor.

Accompanying drawing 20 has been described the confocal microscopy with the KB cell of dendritic macromole processing.

Accompanying drawing 21 has described to use (A) time-histories of dendritic macromole cell growth and (B) dose-dependent inhibition.

Accompanying drawing 22 has described to pass through the growth inhibited of the dendritic macromole of XTT test determination to the KB cell.

Accompanying drawing 23 described to use G5-FITC-FA-MTX and etc. the cell growth inhibited comparison of MTX and the mixture of free FA of molar concentration.

Accompanying drawing 24 has been described the inductive cytostatic reverse that is produced by free FA of G5-FA-MTX-.

Accompanying drawing 25 has been described the stability of dendritic macromole in cell culture medium.

Accompanying drawing 26 has been described the cytotoxicity of dendritic macromole.

Accompanying drawing 27 expression radiolabeled (A) non-targeting and (B) bio distribution of conjugate in the nu/nu mice that has KB xenograft tumor of targeting, be expressed as the percentage ratio of the dendritic macromole of the injected dose that reclaims in every gram organ.

Accompanying drawing 28 expression is to the confocal microscopy analysis from the tumor sample of the frozen section of SCID mice, wherein given described injected in mice: the G5-6-TAMRA of the non-targeting of 10nmol (A) or (B) the G5-FA-6-TAMRA conjugate (B) 15 hours of targeting or (D) 4 days, after this separate tumor.G5-FA-6-TAMRA and G5-6-TAMRA tumor cell specific picked-up relatively is as shown in (C).

Accompanying drawing 29 described put together with G5-FI-FA-MTX and the methotrexate that dissociates (MTX) processing procedure in have the tumor growth of the SCID mice of KB xenograft.

Accompanying drawing 30 has described to have the survival rate of the SCID mice of KB tumor.

Accompanying drawing 31 has been described the synthetic schemes of G5-Ac-AF-RGD.

Accompanying drawing 32 expression G5-Ac-AF-RGD combine with the HUVEC cell.

The combination of accompanying drawing 33 expression G5-Ac-AF-RGD and different cell lines.

The G5-Ac-AF-RGD that accompanying drawing 34 expressions are measured by confocal microscopy combines with the dose dependent of HUVEC cell.

The inhibition of accompanying drawing 35 expressions interpolation free peptide to HUVEC cellular uptake G5-Ac-AF-RGD.

Definition

In order to be conducive to understand the present invention, hereinafter defined a large amount of terms and word:

The combination that term used herein " activating agent " intention has biological related activity or character Thing. Biological related activity for can detect, monitoring or characterising biological reaction or movable biology Reaction or movable relevant activity. Biological related activity includes but not limited to that therapeutic activity (for example changes The ability of the lasting sex change that kind biological health or prevention are relevant with unwanted biological situation), target Active (for example ability of combination or connection biomolecule or compound), active (for example monitoring of monitoring The ability that biological activity progress or monitoring bio composition change), the imaging activity (is for example observed, is Form or the ability of reaction to monitoring bio) and the Marker Identification activity (for example identify some groups of cells The reaction that detects that one-tenth or condition and generation this composition of expression or condition exist). The present invention Activating agent be not limited to these specific way of illustrative example. In fact, can use arbitrarily Useful activating agent comprises the activating agent of sending or destroy biological substance, cosmetic agent etc. In the preferred embodiment of the invention, activating agent or multiple actives and at least a dendroid Big molecule is in conjunction with (for example introduce dendritic macromole, it is first-class that the surface is exposed to dendritic macromole). In certain embodiments of the invention, a kind of dendritic macromole and two or more are " each other Different activating agent is in conjunction with (a kind of dendroid big branch relevant with therapeutic agent with the target agent for example Son). The intention that " differs from one another " is at the activating agent that differs from one another aspect chemical composition and/or the functional group.

Term used herein " nanodevice " intention contains or in conjunction with one or more " activating agent " Little (for example people's naked eyes are invisible) composition. In its simplest form, nanodevice by Combine at least a physical composition that the activating agent of biological functional group (for example therapeutic agent) is provided (for example dendritic macromole) forms. Yet nanodevice can also comprise extra composition (example Such as extra dendritic macromole and/or activating agent). In the preferred embodiment of the invention, The physical composition of nanodevice comprise at least a dendritic macromole and biological functional group by At least a activating agent in conjunction with dendritic macromole provides.

Term used herein " biologically active " intention has natural exist molecular structures, adjusting Or the protein of biochemical function or other bioactive molecule (for example catalysis RNA or little molecule).

A kind of molecule of term used herein " activator " intention, it is being sent out with bioactive molecule During the looks mutual effect, cause biological active component to change (for example promoting), regulate biomolecule and live The property. Activator can comprise protein, nucleic acid, carbohydrate or arbitrarily other in conjunction with biology Bioactive molecule or interactional molecule takes place with it. For example, activator can by directly with RNA polymerase takes place to interact or change Gene Transcription in vitro by transcription factor.

Term used herein " antagonist " or a kind of molecule of " inhibitor " intention, but it is with biological When bioactive molecule takes place to interact, blocking-up or the biologically active of regulating bioactive molecule. Short of money Anti-dose and inhibitor can comprise protein, nucleic acid, carbohydrate or arbitrarily other in conjunction with life Thing bioactive molecule or interactional molecule takes place with it. Inhibitor and antagonist can affect The biological characteristics of whole cell, organ or organism (for example slowing down the inhibitor of tumor growth).

The biologically active of term used herein " adjusting " intention bioactive molecule changes. Regulate Can be increase or the reduction of activity, binding characteristic changes, or living organism property molecule any its The change of its biology, function or immunological characteristic.

Term " gene " intention comprises the nucleic acid that produces polypeptide or the necessary coded sequence of precursor (for example DNA) sequence. Polypeptide can be by complete encoding sequence or by any section of this coded sequence Coded, (for example enzymatic activity, part as long as the required activity of total length or fragment or functional characteristic In conjunction with, signal conduction etc.) kept. This term also comprises code area and the bag of structural gene The sequence of drawing together is positioned at 5 ' and 3 ' end on adjacent position, code area, the distance about 1kb of every end or More than the 1kb, so that this gene is equivalent to the length of full length mRNA. Be positioned at 5 of code area ' also And the sequence that is present on the mRNA is called 5 ' not-translation sequences. Be positioned at that code area 3 ' or the downstream also And the sequence that is present on the mRNA is called 3 ' not-translation sequences. Term " gene " comprises gene CDNA and genome form. The genome form of gene or clone contain be called " introne " or " Interleave the code area of being interrupted by non--coded sequence in district's " or " intervening sequence ". Introne is for transcribing Become the genetic fragment of nRNA (hnRNA); Introne can contain regulating element, such as enhancer. From nuclear or primary transcript, remove or " montage is fallen " introne; Introne is not present in letter thus Make in RNA (mRNA) transcript. MRNA works the sequence of specifying in the newborn polypeptide in translation process Or the effect of amino acid sequence.

Term used herein " nucleic acid molecule encoding ", " dna sequence encoding " and " dna encoding " Intention is along order or the sequence of the deoxyribonucleotide of dna chain. These deoxidation nuclears The order of ribotide has determined along the amino acid sequence of polypeptide (protein) chain. Dna sequence dna by This encoding amino acid sequence.

Antigen part (the example of term used herein " antigenic determinant " intention contact specific antibodies Such as epi-position). When protein or protein fragments are used for giving the host animal immunity, protein Designation area or three-dimensional structure anti-of numerous districts on can the inducing producing specificity conjugated protein Body; These districts or structure are called antigenic determinant. Antigenic determinant can with complete antigen (for example Be used for causing " immunogene " of immune response) the competition binding antibody.

Term " specific binding (specific binding) " or " specific binding (specifically binding) is " when being used in reference to the interaction of antibody and protein or peptide This interaction of intention depends on the ad hoc structure that exists on the protein (antigenic determinant for example Or epi-position); In other words, antibody recognition and binding specificity protein structure rather than general Property ground conjugated protein. For example, if antibody has specificity to epi-position " A ", containing so The egg that contains epi-position A (or free unlabelled A) in " A " of mark and the reaction system of antibody The existence of white matter can reduce the amount of the A of the mark of being combined with antibody.

Term used herein " transgenosis " intention foreign gene is for example by with this foreign gene Import new embryonated egg or body early embryo and be placed in the organism. Term " foreign gene " intention Handle by experiment and import Animal genome and can be included in the gene order of finding in this animal Any nucleic acid (for example gene order) of row is not as long as the gene that imports is present in and natural existence On the identical position of gene.

Term used herein " carrier " is used in reference to dna fragmentation from a kind of cell transfer to another Plant the nucleic acid molecules of cell. Term " carrier (vehicle) " sometimes with " carrier (vector) " Be used interchangeably. Carrier derives from plasmid, bacteriophage or plant or animal virus usually.

Term used herein " expression vector " intention recombinant DNA molecule, it contains required coding The necessary suitable nuclear of coded sequence that is operatively connected in sequence and the expression specific host organism Acid sequence. Express necessary nucleotide sequence in the prokaryotic and generally include promoter, operon (choosing wantonly) and ribosome bind site are usually with other sequence. The known genuine nucleus utilizes Promoter, enhancer also stop and polyadenylation signal.

Term used herein " gene transfer system " intention will comprise the composition of nucleotide sequence and pass Deliver to any means of cell or tissue. For example, gene transfer system includes but not limited to carrier (for example retrovirus, adenovirus, adeno-associated virus and other delivery system based on nucleic acid), The microinjection of naked nucleic acid and based on the delivery system of polymer (for example based on liposome and based on The system of metallic particles). Term used herein " viral gene transfer system " intention comprises favourable (for example complete in the gene transfer system that sample is delivered to the viral element of required cell or tissue The virus of whole virus and modification). Term used herein " adenoviral gene transfer system " intention bag The gene transfer system that contains the virus that belongs to the complete of Adenoviridae or change.

Term used herein " transfection " intention imports eukaryotic with exogenous DNA. Can lead to Cross various modes as known in the art and carry out transfection, comprise calcium phosphate-DNA coprecipitation, DEAE-The transfection of glucan-mediation, 1,5-dimethyl-1,5-phenodiazine 11 methylene gather Methobromide-Transfection, electroporation, microinjection, liposome fusion, fat transfection, the protoplast of mediation melt Close, retroviral infection and biological projectile (biolistics).

Any in vitro culture thing of term used herein " cell culture " intention cell. This art Comprise in the language continuous clone (for example containing the immortalization phenotype), primary cell culture, Finite cell lines (for example not-transformant) and maintain external any other cell mass.

Term used herein " external " intention artificial environment and the process that appears in the artificial environment Or reaction. External environment can be made up of test tube and cell culture, but is not limited to them. Art Language " body in " intention natural surroundings (for example animal or cell) and appear at process in the natural surroundings Or reaction.

Term " test compounds " intention can be used for the treatment of or prevent disease (disease), disease Any chemical entity of sick (illness), disease (sickness) or body function obstacle, Medicine, medicine etc. Test compounds comprises known and potential treatment compound. Can be by making Screen with screening technique of the present invention test compounds is defined as therapeutic agent. " known controls " the intention verified (for example by animal experiment or formerly to the experience of human body administration) for the treatment of compound For in this class treatment or prevention, effectively treating compound.

Term used herein " sample " is with its most widely implication use and comprise environment and life Matter sample. Environmental sample comprises from the material such as soil and this class environment of water. Biological sample Can be animal, comprise the mankind, fluid (for example blood, blood plasma and serum), solid (excrement for example Just), tissue, liquid food (for example milk) and food (for example vegetables).

Term used herein " photosensitizer " transforms when contacting light quantum with " photodynamics dyestuff " intention and reaches the material of excited state.The example of photosensitizer and photodynamics dyestuff include but not limited to photofrin 2, benzoporphyrin ,-tetrahydroxy phenyl chlorin, this alizarinopurpurin stannum (tinetiopurpurin), benzo chlorin copper and other porphyrin class.

Detailed Description Of The Invention

The invention provides be used for the treatment of, the new system and the compositions of analysis and monitoring of diseases (for example cancer).For example, the invention provides system and compositions, its targeting, imaging and sensing Pathophysiology defective provide suitable therapeutic agent based on disease state, and monitoring is to the reaction of the therapeutic agent sent and identify residual disease.In the preferred embodiment of the invention, compositions is enough little to enter patient or experimenter's cell and removes in body being easy to, and does not almost have toxicity under therapeutic dose.

In preferred embodiments, system of the present invention and compositions are used for treatment and/or the monitoring in the cancer therapy process.Yet system of the present invention and compositions are applied to treatment and monitoring various disease states or other physiological conditions, and the present invention is not limited to be used for any concrete morbid state or situation.Specifically be applied to other morbid state of the present invention and include but not limited to cardiovascular disease, viral disease, inflammatory diseases and other proliferative disorders.

In preferred embodiments, the invention provides the 5th generation (G5) polyamidoamine (PAMAM), the dendritic macromole (for example, referring to embodiment 1) of partial acetylation.In other embodiment preferred, the invention provides the method for the multifunctional G5 dendritic macromole of preparation (for example, referring to embodiment 2) and the method (for example, referring to accompanying drawing 1-5) that preparation comprises the dendritic macromole of protected core diamidogen.

The preferred embodiments of the invention provide the compositions that comprises the dendritic macromole of puting together with one or more functional groups, and described functional group includes but not limited to the composition of the specific marker that therapeutic agent, biological monitoring composition, bio-imaging composition, targeted constituent and identification of cell are unusual.Like this, the treatment nanodevice is made by each dendritic macromole, and they respectively carry puts together with dendritic macromole or covalently bound one or more functional groups (for example, referring to embodiment 2 and 6) especially.In preferred embodiments, at least a in the functional group puted together (for example, referring to embodiment 7) by ester bond and dendritic macromole.

Discussion has hereinafter been described each component portion of dendritic macromole and preparation and use their method in certain embodiments of the invention.For design and the application of explaining system of the present invention and compositions, the specific embodiments that part concentrates on the application of compositions in treatment and monitoring breast carcinoma and adenocarcinoma of colon is discussed.These specific embodiments only are used to explain some preferred embodiment of the present invention, but are not used for limiting its scope (for example the compositions and methods of the invention are applied to identify and treat the cell and the tissue of carcinoma of prostate and viral infection).In certain embodiments, dendritic macromole of the present invention is by cell surface part target tumor sexual cell and for example absorb (for example, referring to embodiment 9, accompanying drawing 20) by receptor-mediated endocytosis by tumor cell.In preferred embodiments, imaging component (for example puting together with dendritic macromole of the present invention) makes tumor imaging (for example by using MRI).

In certain embodiments; by therapeutic component is combined with the unstable protection base; such as; for example make cisplatin and photophobic photolabile protecting group be combined with the release that is beneficial to therapeutic agent, wherein said photophobic photolabile protecting group is by being oriented to the emission laser release of those cells of activatory fluorescence color (for example red cell of emission) as mentioned above.Optional therapy equipment (compositions that for example comprises dendritic macromole of the present invention) can also have monitoring target cell or tissue (for example tumor) composition to the reaction of therapy.For example, if the chemotherapeutics of puting together with dendritic macromole of the present invention (for example methotrexate) is induced the cell apoptosis of targeting, the aspartic acid specificity cysteine protease activity of the cell of targeting can be used to activate green fluorescence.This makes the apoptosis sexual cell become orange (red with green combines), and residual cell still keeps red.Induce in the side shoot and apoptotic any normal cell to take place all can follow the infringement green fluorescence.

Find out that as clear from above-mentioned example the application of the present composition helps non-invasive sensing, the signal conduction in cancer and other disease and situation and get involved.Owing to use the concrete scheme of molecular changes in this technical appraisement cancerous cell, so realized the non-invasive sensing of dendritic macromole and can carry out automation application to various tumor phenotypes then.

I. dendritic macromole

In preferred embodiments, compositions of the present invention comprises dendritic macromole (for example, referring to accompanying drawing 1-5 and embodiment 2).Extensively described dendritic (referring to, Tomalia, Advanced Materials 6:529 (1994); Angew, Chem.Int.Ed.Engl., 29:138 (1990); These documents intactly are incorporated herein by reference).Dendritic can be synthesized the chondritic determined of general diameter in the 1-20 nanometer.Shown that preparation has the method (accompanying drawing 1-5) of the G5PAMAM dendritic macromole of protected core.In certain embodiments, protected core diamidogen is NH2-CH2-CH2-NHPG.Molecular weight and end group quantity are index increase (for example, referring to accompanying drawing 9) as the function in the generation (number of plies amount) of polymer.Can be based on the synthetic dissimilar dendritic macromole (for example, referring to accompanying drawing 1-5) of the core texture that starts polymerization process.

The core texture of dendritic macromole has been described several features of molecule, such as overall shape, density and surface functionality (Chem.Int.Ed.Engl. such as Tomalia, 29:5305 (1990)).Spherical dendritic macromole can have ammonia as trivalent initiator core or ethylenediamine (EDA) as trivalent initiator core (for example, referring to accompanying drawing 9).At present the lopstick dendritic macromolecules of describing J.Am.Chem.Soc. such as (, 120:2678 (1998)) Yin uses the polymine linear core (for example core is long more, and then club is long more) of different length.Dendritic macromole for by kilogram quantities be purchased and be used for present good preparation method (GMP) generation of biotechnology applications.

Can characterize dendritic macromole by many technology, include but not limited to the electrospray ionization mass spectrum, ¹³The C nuclear magnetic resonance spectrometry, ¹H nuclear magnetic resonance spectrometry (for example, referring to embodiment 5, accompanying drawing 10 (A) and embodiment 7, accompanying drawing 14), high performance liquid chromatography (for example, referring to embodiment 5, accompanying drawing 10 (B); With embodiment 6, accompanying drawing 13), use size exclusion chromatography (for example, referring to embodiment 4, accompanying drawing 8), ultraviolet spectrophotometry (for example, referring to embodiment 8, accompanying drawing 17), capillary electrophoresis and the gel electrophoresis of multiple angle laser light scattering.These tests have guaranteed that the quality control that polymer group's uniformity and the dendritic macromole that monitoring is used for using in GMP application and the body prepare is important.

The method and composition that is used to produce dendritic macromole has been described in numerous United States Patent (USP)s.Provided some the embodiment in these patents below, so that provide to being used for the description of some dendrimer composition of the present invention, but, these should be understood and only the present invention can be used for for explanatory embodiment and numerous other similar dendrimer composition.

U.S. Pat 4,507,466, in U.S. Pat 4,558,120, U.S. Pat 4,568,737 and the U.S. Pat 4,587,329 each self-described preparation have method greater than the fine and close star polymer of the terminal density of conventional star polymer.These polymer have greater than/be higher than the homogeneous reaction of conventional star polymer, promptly the 3rd generation fine and close star polymer.The character (amidoamine) of amidoamines dendritic macromole and the 3-dimension molecular diameter of this dendritic macromole have been further described in these patents.

U.S. Pat 4,631 has been described the polymer of hydrolysis-stable in 337.U.S. Pat 4,694 has been described the clavate dendritic macromole in 064.U.S. Pat 4,713 has been described fine and close star polymer and the application in characterizing the surface that virus, antibacterial and protein comprises enzyme thereof in 975.The fine and close star polymer of bridging is described in U.S. Pat 4,737, in 550.U.S. Pat 4,857,599 and U.S. Pat 4,871,779 in the sensitization star polymer on the immobilization core of using as ion exchange resin, chelate resin and the preparation method of this base polymer have been described.

U.S. Pat 5,338,532 relate to star explosion shape (starburst) conjugate of the unitary dendritic macromole that combines at least a agricultural of carrying, medicine or other material.This patent has been described the delivery apparatus of per unit polymer, controlled delivery, targeted delivery and/or multiple class middle and high concentration belongings matter, described multiple class such as, for example medicine antibiotic, general and concrete toxin, metal ion, radionuclide, signal generator, antibody, interleukin, hormone, interferon, virus, viral fragment, insecticide and antimicrobial.

U.S. Pat 6,471, the dendritic macromole complex that comprises first kind and second kind covalently bound dendritic macromole has been described in 968, wherein first kind of dendritic macromole comprises first kind of activating agent, and second kind of dendritic macromole comprises second kind of activating agent, wherein first kind of dendritic macromole is different from second kind of dendritic macromole, and wherein first kind of activating agent is different from second kind of activating agent.

Other useful dendritic macromole based composition is described in U.S. Pat 5,387, and 617, U.S. Pat 5,393,797 and U.S. Pat 5,393, in 795, wherein by modifying fine and close star polymer with the hydrophobic group end-blocking that hydrophobic shell can be provided.U.S. Pat 5,527 has disclosed the application of amino-terminated dendritic macromole in antibody conjugates in 524.

Dendritic macromole is described in U.S. Pat 5,560 as the application of carriers of metal ions, in 929.U.S. Pat 5,773 has disclosed non-crosslinked multibranched polymers of comb shape explosion shape structure and preparation method thereof in 527.U.S. Pat 5,631 has been described the method that generates the high molecular multibranched polymers in 329, carries out through the following steps: form first group of branched polymer by branch; With the core grafting; Make first group of branched polymer deprotection, form then second group because of the protected branched polymer of branch and with core grafting with first group of branched polymer etc.

U.S. Pat 5,902 has been described the dendritic macromole reticulated structure that contains lipotropy organosiloxane and hydrophilic polyanicloamine nanscopic domains in 863.This reticulated structure is by having the inner and outer field copolymerization dendritic macromole of the organosilicon precursor preparation of PAMAM (hydrophilic) or polypropylene imines.These dendritic macromoles have controlled size, shape and spatial distribution.They are to have the complex that can be used for special-purpose film, protective finish, contain organic metal or inorganic additive, and skin patch is sent, absorbent, chromatography personal care product and agricultural product have an outer field hydrophobicity dendritic macromole of organosilicon.

U.S. Pat 5,795 has been described the application of dendritic macromole as the adjuvant of influenza antigens in 582.The application of dendritic macromole has produced the antibody titer level of the antigen dose that has reduction.U.S. Pat 5,898,005 and U.S. Pat 5,861,319 in described be used for the determination and analysis substrate concentration specific immunity in conjunction with test.U.S. Pat 5,661, the polynucleotide delivery system that the oneself's assembling that comprises the dendritic macromole polycation is provided in 025 is so that help the detailed description of nucleotide delivery to target site.This patent provides external polynucleotide has been imported eukaryotic method, comprise make a kind of compositions of cells contacting, said composition comprise polynucleotide and with the non-covalent link coupled dendritic macromole polyeation of these polynucleotide.

Verified in advance dendritic macromole-antibody conjugates is used Clin.Chem. such as (, 40:1845 (1994)) Singh, is being produced dendritic macromole-chelating agen-antibody construct and in the application of research and development boronation dendritic macromole-antibody conjugates (being used for neutron capture therapy) in in-vitro diagnosis; In these chemical compounds of back each can be used as cancer therapeutic agent (Bioorg.Med.Chem.Lett. such as Wu, 4:449 (1994); Magn.Reson.Med.31:1 such as Wiener (1994); Bio conjugate Chem.5:58 (1994) such as Barth; With Barth etc.).

Some ((1994) such as Wu etc. (1994) and Wiener, document is the same) in these conjugates in the nuclear magnetic resonance of tumor, have also been used.The structure that obtains from this work has write down when carrying out vivo medicine-feeding, and antibody can make dendritic macromole-bonded therapeutic agent be oriented to have antigenic tumor.Enter cell with also confirming the dendritic macromole antigenic specificity and carry chemotherapeutics or the genetic therapy agent.Especially, studies show that the cisplatin that is encapsulated in the dendritic macromole polymer has increased effect and the cisplatin (Duncan and Malik, Control Rel.Bioact.Mater.23:105 (1996)) that provides alternate manner to send is provided toxicity.

Dendritic macromole can also be puted together the verified cell that enters with fluorescent dye or molecule indicateing arm.Can in cell, detect according to the mode compatible then with the sensor apparatus of estimating physiological change in the cell they Anal.Chem.69:990 (1997) such as () Baker.Finally, the big branch of dendroid is configured to differential block copolymer, wherein can digests the outer part (Urdea and Hom, Science 261:534 (1993)) of molecule with enzyme or photoinduced catalysis.This can controlling polymers degraded so that discharge therapeutic agent and can provide mechanism for the external trigger that discharges therapeutic agent at disease location.

In certain embodiments, the invention provides dendritic macromole, be formed with one or more functional groups of respectively carrying concrete degree of functionality (for example, referring to embodiment 7 and 8, accompanying drawing 14 and 15) in the wherein single dendritic macromole.For example; preferred compositions of the present invention comprises the 5th generation (G5) the PAMAM dendritic macromole of partial acetylation; this dendritic macromole further comprises therapeutic agent, targeting agent and preparation; wherein therapeutic agent comprises methotrexate; the targeting agent comprises folic acid; and preparation comprises Fluorescein isothiocyanate (for example, referring to embodiment 7 and 8).Thus, the invention provides single multifunctional dendritic macromole.In certain embodiments, on single dendritic macromole, formed any one (for example therapeutic agent) in the above-mentioned functional group of a plurality of copies.For example, in certain embodiments, single dendritic macromole comprises the single functional group (for example therapeutic agent, such as methotrexate) of 2-100 copy.In other embodiment preferred; the invention provides the 5th generation (G5) the PAMAM dendritic macromole of partial acetylation; this dendritic macromole further comprises therapeutic agent, targeting agent and preparation, and wherein the targeting agent comprises RGD peptide (for example, referring to embodiment 14).In certain embodiments, the invention provides the 5th generation (G5) the PAMAM dendritic macromole of partial acetylation, this dendritic macromole comprises therapeutic agent, targeting agent and preparation, and wherein therapeutic agent comprises tritium (for example, referring to embodiment 13).

II. therapeutic agent

Various therapeutic agents are applied to the present invention.Therefore, the present invention is not limited to the type of the therapeutic agent that can put together with dendritic macromole of the present invention.Can use method of the present invention, system and compositions to send any therapeutic agent in conjunction with dendritic macromole.In order to explain sending of therapeutic agent, discussion hereinafter mainly concentrates on methotrexate, cisplatin and taxol sending in the treatment cancer.Various photodynamic therapy chemical compounds and various Antimicrobe compound also have been discussed.

I. methotrexate, cisplatin and taxol

The cytotoxicity of methotrexate depends on persistent period of keeping level in the threshold value born of the same parents (Cancer Res 58,5749 (1998) such as Levasseur; Goldman ﹠amp; Matherly, Pharmacol Ther 28,77 (1985)).Cell contains the DHFR of high concentration, and has interrupted the DHFR activity fully, need be higher than the anti-folate agent level (Sierrra of 6 orders of magnitude of Ki of DHFR; Goldman, Seminars in Oncology 26,11 (1999)).In addition, being lower than 5% enzymatic activity is enough (White for complete cell enzymatic functions; Goldman, Biol Chem 256,5722 (1981)).Cisplatin and taxol have fully determine in tumor cell cell death inducing effect (for example, referring to Proc.Natl.Acad.Sci. such as Lanni, 94:9679 (1997); Cancer Research57:5107 (1997) such as Tortora; With Brit.J.Cancer 77:1378 (1998) such as Zaffaroni).Yet, use these and other chemotherapeutics treatment to be difficult under the situation of the overt toxicity that does not cause, carry out.At present these activating agents of using generally are insoluble in water, and toxicity obviously and give under the dosage that influences normal cell and diseased cells.For example, one of optimal anticancer compound of discovery paclitaxel (taxol) is insoluble in water.

Paclitaxel has demonstrated splendid anti-tumor activity in various tumor models, such as B16 melanoma, L1210 leukemia, MX-1 mammary neoplasms and CS-1 colon tumor xenograft.Yet administration has proposed a difficult problem to paclitaxel to human body at poorly water-soluble.Therefore, the formulation for paclitaxel that uses at present needs the cremaphor solubilize drugs.Human clinical's dosage range is at 200-500mg.This dosage is dissolved in 1: 1 ethanol: cremaphor solution and be diluted to the 1 up-flow body that gives through intravenous.The cremaphor that uses at present is GREMAPHOR GS32.By being dissolved in the cremaphor mixture and diluting it is given by infusion with the aqueous vehicle of large volume.Directly administration (for example subcutaneous) produces local toxicity and low-level activity.Therefore, for these chemotherapeutics, there is demand in more effective and effective delivery system.

The present invention has overcome these difficult problems by the method and composition that is provided for specifically passing medicine.The present invention also provides the combination (for example two or more different therapeutic agents) that gives activating agent to produce the ability of additive effect.The application of multiple actives can be used for the drug resistance that resist the disease produces any single-activity agent.For example, reported the drug resistance (Molecular Cell.2:581 (1998) such as Yu) of some cancer to single medicine (taxol).The verified methotrexate of puting together with dendritic macromole of the experiment of in R﹠D process of the present invention, carrying out can effectively kill and wound cancerous cell (referring to embodiment 10, accompanying drawing 21 and 22 and embodiment 12, accompanying drawing 26).

The present invention also provides the treatment opportunity of success of monitoring after methotrexate and/or cisplatin and/or taxol are delivered to the experimenter.For example, the sign (Gibb, Gynecologic Oncology65:13 (1997)) that the external evoked apoptotic ability of these medicines is reported to effect in the body will be measured.Therefore, except that one of these medicines (or other therapeutic agent), wherein two kinds or all targeted delivery can also be monitored the effectiveness of the technology evaluation therapy of apoptosis-inducing by the present invention effective antitumor therapy to be provided and to reduce the toxicity.Importantly, these therapies have activity to extensive tumor type, include but not limited to breast carcinoma and colon cancer (Eur.J.Cancer 31A:2341 (1995) such as Akutsu).

Although three species specificity activating agents have been described in above-mentioned discussion, the conventional any activating agent (for example medicine) that is used for the cancer therapy content all can be applicable to the present invention.Treating according to the present invention in the process of cancer, the therapeutic component of dendritic macromole can inclusion compound, includes but not limited to amycin, 5-fluorouracil, etoposide, camptothecine, actinomycin D, ametycin, more preferably cisplatin.Can prepare this activating agent and by its as described herein and immunotherapeutic agent being merged as uses such as the therapeutic combination of merging or test kits.

In certain embodiments of the invention, the expection dendritic macromole comprises one or more activating agents, and their direct interconnection nucleic acid (for example DNA) is so that help DNA infringement, thereby produces the synergistic antineoplastic agent that has of the present invention.Can use such as this class activating agent of cisplatin and other DNA alkylating agent.Cisplatin has been widely used in treating cancer, and wherein being used for the clinical effectiveness dosage of using is 20mg/M ², continuing 5 days, per 3 weeks carry out 1 time, amount to 3 courses of treatment.Can send dendritic macromole by the method for any appropriate, include but not limited in intravenous, subcutaneous, the tumor, intraperitoneal or part (for example being delivered to mucomembranous surface).

The activating agent of infringement DNA also comprises the chemical compound that disturbs dna replication dna, mitosis and chromosome separation.This based chemotherapy chemical compound comprises: amycin is also referred to as doxorubicin; Etoposide; Verapamil; Podophyllotoxin etc.Give widely used these chemical compounds in the clinical settings of treatment tumor by the intravenous bolus injection, with regard to amycin, dosage range is 25-75Mg/M ², 21 days at interval, with regard to etoposide, the oral dual dosage of intravenous or intravenous was 35-50Mg/M ²

Destroy that nucleic acid precursor and subunit activating agent synthetic and degree of accuracy also causes the DNA infringement and as the chemotherapeutics among the present invention.A large amount of nucleic acid precursors been have have been researched and developed.Useful especially activating agent for having carried out extensive testing and be used to obtain.Like this, preferentially utilized, make this activating agent be used in particular for the target tumor sexual cell by the tumprigenicity tissue such as this class activating agent of 5-fluorouracil (5-FU).The dosage of sending can be 3-15mg/kg/ days scope, and but, other dosage can obviously change according to different factors, comprises that the stage, cell of disease are to the compliance of therapy, to the amount of activating agent tolerance etc.

Be applied to anticancer therapeutic agent of the present invention and be easy to introduce the dendritic macromole structure for those, or otherwise just with the bonded therapeutic agent of dendritic macromole structure, make them to be sent into experimenter, tissue or cell, and the degree of accuracy of its antitumaous effect can not lost.For the more detailed description cancer therapeutic agent, such as platinum complexes, verapamil, podophyllotoxin, carboplatin, procarbazine, chlormethine, cyclophosphamide, camptothecine, ifosfamide, melphalan, chlorambucil, busulfan (bisulfan), nitroso ureas (nitrosurea), amycin, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicomycin, bleomycin, etoposide (VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastine and methotrexate and other similar anticarcinogen, those skilled in the art have consulted many illustrative handbooks, include but not limited to " Pharmaceutical Basis of Therapeutics " ninthedition of Physician ' s Desk reference and Goodman and Gilman, Eds.Hardman etc. 1996.

In certain embodiments, preferably make connection base that medicine is combined with dendritic macromole with the light cleavable.For example, being applied to several Heterobifunctional light cleavable of the present invention connects base and describes Bio conjugate Chem. such as (, 9:143 (1998)) Ottl by Ottl etc.These connect, and base can be for water or organic soluble.They contain activatory ester, this ester can with can with amine or the alcohols and the epoxide reaction of sulfydryl reaction.Be 3 between two kinds of groups, 4-dimethoxy 6-nitrobenzophenone photoisomerization group, this group discharge the amine or the alcohol of complete form when contact black light (365nm).Therefore, when using this class connection base to be connected with the present composition, by making target area contact black light, therapeutic agent can be released with biological activity or activable form.

In a preferred embodiment, methotrexate is puted together (referring to for example embodiment 7) by ester bond and dendritic macromole.In a typical embodiment, the alcohol radical of taxol is connected the Acibenzolar reaction of base with organic soluble.This product thus with the part mercaptan surface reaction of suitable dendritic macromole (the primary amine class of dendritic macromole can by partly being changed into the group that contains sulfydryl) with the 2-iminothiolano reaction of inferior stoichiometric amount.With regard to cisplatin, the amino of medicine and the water-soluble form reaction that is connected base.If amino reaction not exclusively, can use the active analogue thereof that contains primary amino radical of cisplatin so, such as Pt (II) sulfadiazine dichloride (Inorg.Chim.Acta 80:99 (1983) such as Pasani and Abel etc., Eur.J.Cancer 9:4 (1973)).Therefore, the medicine non-activity of puting together and can not damage normal cell.When conjugate was arranged in tumor cell, its contact is the laser of near-UV wavelength suitably, thereby cause active medicine to be released into cell.

Similarly, in other embodiments of the present invention, the amino that makes cisplatin (or its analog) with have hydrophobic smooth cleavable protecting group, be connected (Pillai, V.N.R.Synthesis:1-26 (1980)) such as 2-nitro benzyloxycarbonyl group.Owing to connected this hydrophobic group, so medicine is loaded into the hydrophobic pocket of PAMAM dendritic macromole and very preferentially by its reservation (for example, referring to Pharm.Sci. such as Esfand, 2:157 (1996)), isolates with water environment.When contact near-during LV light (about 365nm), hydrophobic group is cleaved, discharges complete medicine.Because medicine is certainly as hydrophilic, so it diffuses out and enter tumor cell from dendritic macromole, it can start apoptosis there.

Connect base as the attachable alternatives that connects base of light for the enzyme cleavable.Verified a large amount of cleavable connects base and is effective antitumour conjugate and can be by making cancer therapeutic agent, the water-soluble polymer that is connected base such as doxorubicin with the peptide that has suitable weak point is in conjunction with (for example preparing, referring to Clin.Cancer Res. such as Vasey, 5:83 (1999)).Connecting base is stable in outside, but in case in cell just by the thiol protease cracking.In a preferred embodiment, use PK1.Connect the alternative of base strategy as the light cleavable, can use the degradable connection base of enzyme, such as Gly-Phe-Leu-Gly.

The present invention is not limited to the character of treatment technology.For example, be applied to other conjugate of the present invention and include but not limited to use the boron dredger of puting together (the Bio conjugate Chem. such as Capala that is used for BNCT, 7:7 (1996)), use radiosiotope and combine with nanodevice such as this toxoid of Ricin.

Ii. photodynamic therapy

The photodynamic therapy therapeutic agent also can be as the therapeutic agent among the present invention.In certain embodiments, irradiation the present invention contains the dendrimer composition of photodynamic compound, cause singlet oxygen and free-radical generating, they diffuse out from no fiber radiation effect thing and biological target (for example tumor cell or bacterial cell) are worked.Some preferred photodynamic compound includes but not limited to that those participate in the photochemically reactive chemical compound of II type:

→

PS+hv PS ^*(1)

→

PS ^*(1) PS ^*(3)

→

PS ^*(3)+O ₂ PS+ ^*O ₂

→

^*O ₂+ T cytotoxicity

PS=photosensitizing agents wherein, the singletstate of the PS of PS* (1)=excite, the triplet state of the PS of PS* (3)=excite, hv=light quantum, * O ₂The singletstate of=the oxygen that excites, and T=biological target.Be used for other photodynamic compound of the present invention and comprise that those pass through different mechanisms that non-singlet oxygen produces and produce Cytotoxic chemical compound (copper benzochlorin for example, Selman, Deng Photochem.Photobiol., 57:681-85 (1993) is incorporated herein by reference).The example that is applied to photodynamic compound of the present invention includes but not limited to phytochrome 2, phthalocyanine (phtalocyanins) (for example, referring to Photochem.Photobiol. such as Brasseur, 47:705-11 (1988)), benzoporphyrin, tetrahydroxy phenyl porphyrin class, naphtalocyanines (for example, referring to Firey and Rodgers, Photochem.Photobiol., 45:535-38 (1987)), sapphyrins (Proc.SPIE such as Sessler, 1426:318-29 (1991)), porphin ketone (porphinones) (Proc.SPIE such as Chang, 1203:281-86 (1990)), this alizarinopurpurin stannum (tin etiopurpurin), the porphyrin class that ether replaces (Photochem.Photobiol. such as Pandey, 53:65-72 (1991)) and cationic dye, such as fen  piperazine class (for example, referring to SPIE Proc. such as Cincotta, 1203:202-10 (1990)).

Iii. antimicrobial therapy agent

Antimicrobial therapy agent of the present invention also can be as the therapeutic agent among the present invention.Can use and can kill and wound, suppress, or otherwise be exactly any activating agent that weakens microorganism biological body function, and concern has the active any activating agent of this class.Antimicrobial includes but not limited to natural and synthetic antibiotic, antibody, Profilin, antisensenucleic acids, film destroy agent etc., can be with them separately or unite use.In fact, the antibiotic of any type be can use, antibacterial, antiviral agent, antifungal etc. included but not limited to.

III. labelling identifier

In certain embodiments, nanodevice of the present invention contains one or more by marked member (" labelling ") activation or interactional labelling identifier can take place with it.In preferred embodiments, the labelling identifier is the antibody of specificity incorporation of markings, preferred monoclonal antibody (for example the cell to targeting has specific cell-specific molecule).

In certain embodiments of the invention, identified tumor cell.Tumor cell has various labellings, comprises the cancer specific antigen of determining expression, such as the p53 of the Muc1 in the breast carcinoma, HER-2 and sudden change.They work as the specific marker of cancer, and the amount in breast carcinoma is 30% (HER-2)-70% (p53 of sudden change).In a preferred embodiment, dendritic macromole of the present invention comprises specificity in conjunction with the monoclonal antibody that is present in the p53 mutant form in the breast carcinoma.

In certain embodiments of the invention, identified the cancerous cell of expressing susceptibility gene.For example, in certain embodiments, there are two kinds of breast carcinoma susceptibility genies: the BRCA2 on BRCA1 on the chromosome 17 and the chromosome 13 as the breast carcinoma specific marker.When individuality carries sudden change among BRCA1 or the BRCA2, during the risk of mammary gland or ovarian cancer increased when they were diagnosed as some point that is in the life.These genes participate in repairing the inductive fracture of radiation in the double-stranded DNA.Think that the sudden change among BRCA1 or the BRCA2 may make this mechanism forfeiture, cause the mistake of DNA in repairing more and finally cause the cancer growth.

In addition, the expression of a large amount of different cell surface receptors is as the target of nanodevice combination and absorption.This receptoroid includes but not limited to EGF receptor, folacin receptor, FGR receptor 2 etc.

In certain embodiments of the invention, marked member is changed in relevant with chromosomal abnormality (abborations) gene expression.For example, Burkitt lymphoma causes because of the chromosome translocation that relates to the Myc gene.Chromosome translocation intention chromosome breakage makes the chromosomal part of it and other be connected.Traditional Burkitt lymphoma in the Burkitt lymphoma relates to chromosome 8, i.e. the site of Myc gene.The change of this Myc expression pattern destroys its common function in growth of control cell and propagation thus.

In other embodiments, the gene expression relevant with the contact cancer is accredited as marked member.Known two kinds of key genes relate to colon cancer: the MLH1 on MSH2 on the chromosome 2 and the chromosome 3.Generally speaking, the protein of these genes helps DNA plerosis to duplicate the middle mistake that forms.If MSH2 and MLH1 protein mutant, the mistake in duplicating so still can't be repaired, thereby produces impaired DNA and colon cancer.The in the past few years known MEN1 gene that relates to MEN syndrome is found on chromosome 11, in 1997 it is mapped more subtly, and is used as the labelling of this class cancer.In a preferred embodiment of the invention, make gene to the protein of the change that detects or expression have specific antibody and nanodevice of the present invention is compound.

In another embodiment, adenocarcinoma of colon has been determined the expression of the p53 of CEA and sudden change, and the two is well-verified tumor marker.P53 sudden change in some of these cells is similar to observed sudden change in some breast cancer cell, and can have the p53 sensing composition (promptly at the assembling nanodevice, the dendritic macromole that comprises the same tag identifier can be used for every kind of cancer types) that is used between these cancers two kinds of nanodevices separately.Can use the cell line that in nude mice, produces tumor to study colon and breast cancer cell reliably, so that optimization in animal and sign.

Can clearly be seen that from above-mentioned discussion have many different tumor markers of the present invention that are applied to, wherein some has specificity to the cancer of particular type, and other belongs to its aspect, source accidental (promiscuous).The present invention is not limited to any specific tumors labelling or other disease specific labelling arbitrarily.For example, the tumor suppressor thing as labelling of the present invention includes but not limited to p53, Mucl, CEA, p16, p21, p27, CCAM, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-1, MEN-II, p73, VHL, FCC and MCC.

IV. biology imaging component

In certain embodiments of the invention, nanodevice comprises at least a nanoscopic structural unit that is easy to imaging based on dendritic macromole.The present invention is not limited to use the character of imaging component.In certain embodiments of the invention, image-forming assembly comprises the finishing of quantum dot (for example, referring to Chan and Nie, Science 281:2016 (1998)), such as with the link coupled zinc sulfide of biomolecule-end capped cadmium selenide (Sooklal, Adv.Mater., 10:1083 (1998)).

Yet, in preferred embodiments, image-forming assembly comprises according to the dendritic macromole of the production of " nano-complex " notion (Proc.of ACS PMSE 77:118 (1997) and Balogh and Tomalia such as Balogh, J.Am.Che.Soc., 120:7355 (1998)).In these embodiments, produce dendritic macromole by reactive encapsulation, wherein reactant by the pre-structure of dendritic macromole template and be fixed in the polymer molecule by second kind of reactant subsequently/on.Measure size, shape, size distribution and the surface functionality of these nanoparticles and control by dendritic macromole.These materials have host's dissolubility and the compatibility and have the optics or the physiological property of enclosed molecule (guest molecule) (promptly allowing the molecule of imaging).Although the dendritic macromole host can change according to the difference of medium, load different chemical compounds and different object concentration levels can for the dendritic macromole host.Complex and compositions can relate to the application of various metals or other inorganic material.The high electron density of these materials has obviously been simplified the imaging by electron microscopy and dependent scattering technology.In addition, the characteristic of inorganic atoms has been introduced and has been had or do not exist the new and characteristic that can measure that is used for imaging under the interference biomaterial.In certain embodiments of the invention, gold, silver, cobalt, iron atom/molecule and/or inorganic dyestuff molecule with encapsulation, go into dendritic macromole so that, but, can use any materials that helps imaging or detection such as the fluorescein encapsulation as nanoscopic complex labelling/tracer.In a preferred embodiment, preparation is a Fluorescein isothiocyanate.

In certain embodiments of the invention, imaging is based on to the passive of the local difference of density in the physical characteristic of selection of the complex material of research or observed result initiatively.These differences may be because of due to difformity (for example mass density that detects by the atomic force microscope inspection), the composition (for example radiopacity that detects by X ray) that changes, different light emission (for example fluorochrome that detects by spectrophotography), different diffraction (for example electron beam that detects by TEM), correlated absorption (for example light that detects by optical means) or the special-purpose radiation emission (for example isotope method) etc.Therefore, the technology of observed characteristic and use is depended in the quality of imaging and sensitivity.The imaging technique of cancerous cell must provide the sensitivity to enough levels of the selection cell of observing little local concentration.Initial cancer labelling is identified, and high selectivity (i.e. the high degree of specificity identification that provides by suitable targeting) and the highest possible sensitivity are provided.

A. nuclear magnetic resonance

In case the nanodevice of targeting is in conjunction with (or having taken in) tumor cell, then one or more assemblies on this device are used to make its position imaging.As the biomedical imaging agent, for nuclear magnetic resonance (MRI), this may be the most noticeable (for example, referring to Mag.Reson.Med.31:1 such as Wiener (1994) to dendritic macromole; Use the example of PAMAM dendritic macromole).Generally by making the paramagnetic ion of chelating, (Gd (III)-DTPA) puts together formation with the water solublity dendritic macromole to these reagent such as Gd (III)-diethylene-triamine pentaacetic acid.Operable other paramagnetic ion includes but not limited to gadolinium, manganese, copper, chromium, ferrum, cobalt, erbium, nickel, europium, technetium, indium, samarium, dysprosium, ruthenium, ytterbium, yttrium and holmium particle and combination thereof in the context of the invention.In certain embodiments of the invention, dendritic macromole is also with the targeting group, put together such as epidermal growth factor (EGF), so that form the conjugate (for example with regard to the tumor cell of expressing EGF, EGFR with regard to) of specificity in conjunction with required cell type.In an embodiment preferred of the present invention, as described in Wiener, the isothiocyanate of DTPA by DTPA combines Mag.Reson.Med.31:1 (1994) such as () Wiener with dendritic macromole.

Dendritic macromole MRI reagent is effective especially because of polyvalency, size and the structure of dendritic macromole, produces the molecule with big proton relaxation reinforcement, high molecular weight reactive (relaxivity) and the high paramagnetic ion concentration on target site.Dendritic macromole gadolinium contrast agent even be used to use Dynamic MRI to be used for distinguishing benign and malignant breast tumor is based on the vascular system of back one type tumor image more fine and close (Ivest.Rad.31:26 (1996) such as Adam).Therefore, MRI provides the present invention useful especially imaging system.

B. microscope imaging

Traditionally at the external static structure microscope imaging that carries out cancerous cell and tissue of patient.Organize bioptic traditional histology that meticulous illustrative example is provided, and turned out to be the strong auxiliary of cancerous diagnose and treatment.After the taking-up, sample is thinly sliced (for example less than 40 microns), dyeing, fixing and inspection by the pathologist.If the acquisition image, so they modal be that 2-D transmits bright field projection image.Special-purpose dyestuff is used to provide the selectivity contrast, and does not exist hardly undyed organizing, and the evaluation to the abnormal cell composition is provided.Such as the auxiliary analysis that uses a computer in the nuclear ploidy is measured the subcellular structure feature is carried out quantitatively easily obscuring because of histology's background disappearance usually, this is owing to sample and all 3-D poor informations.Although there is the limitation of quiescent imaging means, can identify that the neoplasia in the biopsy tissue is very important.In addition, its application is generally the key factor in chemotherapy, operation technique and the radiotherapy combination that determines to carry out invasive and danger, and serious collateral tissue infringement, complication and even death are followed in chemotherapy, operation technique and radiotherapy usually.

Nanodevice of the present invention can carry out the functional microscope imaging of tumor and the method for improvement is provided for imaging.It is interior, external and stripped that these methods are applied to body.For example, in one embodiment of the invention, design dendritic macromole of the present invention so that at contact light time emission light or other detectable signal.Although less than the optical resolution limit of microscopy technology, they become self-luminous when being excited and are easy to use optical technology to observe and can measure the dendritic macromole of labelling in shape.In certain embodiments of the invention, the sensing biological sensor in the microscope comprises using and tunablely excites and launch filter and microwave source SPEI 2678:200 (1997) such as () Farkas.In preparation is present in embodiment in the darker tissue, use wavelength (for example, referring to Cel Mol.Biol.44:29 (1998) such as Lester) long in the near-infrared (NMR).Used dendritic macromole bio-sensing feeler spline structure to confirm bio-sensing J.Phys.Chem. such as (, 101:6318 (1997)) Shortreed of the dendritic macromole among near-IR.Be used for biosensor of the present invention and include but not limited to fluorescent dye and molecule indicateing arm.

In certain embodiments of the invention, the function of use imaging technique carries out in-vivo imaging.Functional imaging be with static structure imaging complementarity and may be for than the more virtuous technology of static structure imaging.The known function imaging preferably is applied on macroscopic scale, and wherein example comprises functional magnetic resonance imaging (fMRI) and positron emission tomography (PET).Yet, also carry out functional microscope imaging and be applied in the living tissue body and the analysis of exsomatizing.Functional microscope imaging is that 3-D imaging, the multispectral volume in 3-D space are arranged and effective combination of sampling temporarily: in brief, be a kind of 3-D spectromicroscope film cyclical patterns.Meaningfully, cell and organize autofluorescence.When being excited, provide numerous basic 3-D structures need under the situation of no specific marker, characterize several cell component (for example nuclear) by several wavelength.The skew ray irradiation also is used to gather structural information and uses usually.Opposite with the micropatterning (microimaging) of structure spectra, the spectrum micropatterning can be used to make the biosensor of physiological signal positioning action in cell or tissue.For example, in certain embodiments of the invention, the dendritic macromole of the present invention that comprises biosensor is used to make the receptor family imaging of up regulation, such as folate or EGF class.In this class embodiment, the functional living being sensing relates to detection and carcinogenesis or malignant tumor even thus relevant physically different of its commitment.Can use the compositions and methods of the invention to make many physiological condition imagings, include but not limited to detect pH, oxygen concentration, the Ca of nanoscopic dendritic macromole biosensor ²⁺Concentration and other physiology analyte of interest.

V. monitor composition biology

The biological detection of nanodevice of the present invention or sensing composition are for the composition by the particular responses in the inductive tumor cell of activating agent (for example therapeutic agent that provides by the therapeutic component in the nanodevice) can be provided.Although the present invention is not limited to any specific monitoring system, the present invention explains by the method and composition that is used to monitor cancer.In the preferred embodiment of the invention, apoptosis in the reagent inducing cell and monitoring relate to the detection apoptosis.In specific embodiment, the monitoring composition be when apoptosis takes place under specific wavelength fluorescigenic reagent.For example, in a preferred embodiment, the green fluorescence in the active activation monitoring of the aspartic acid specificity cysteine protease composition.It is orange by the result's of the specific markers targeting with red-label apoptosis cancerous cell change to be transformed into red conduct, and residual cancerous cell still is red.If exist, induced so and carried out apoptotic normal cell (for example by collateral damage) meeting green-emitting fluorescence.

In these embodiments, fluorophor is used to monitor composition such as fluorescein.Fluorescein is easy to by available from Molecular Probes, the isothiocyanic acid ester derivant of Inc. and dendritic macromole surface combination.This allows by Laser Scanning Confocal Microscope, uses cell to make the nanodevice imaging.Preferably by use fluorescence peptide zymolyte to nanodevice effectiveness carry out sensing.For example, the apoptosis by therapeutic-induced causes peptidase aspartic acid specificity cysteine protease-1 (ICE) to produce.Calbiochem has sold the peptide substrates of the release fluorescence part that is used for this kind of enzyme in a large number.Being used for useful especially peptide of the present invention is:

MCA-Tyr-Glu-Val-Asp-Gly-Trp-Lys-(DNP)-NH ₂(SEQ?ID?NO：1)

Wherein MCA is (ayapanin-4-yl) acetyl group, and DNP is 2,4-dinitrophenyl (J.Biol.Chem. such as Talanian, 272:9677 (1997)).In this peptide, the MCA group has the fluorescence that significantly weakens, and this is can shift (FRET) due to the DNP group because of fluorescence resonance.When the peptide between enzymatic lysis aspartic acid and the glycine residue, MCA is separated with DNP, and the MCA group sends green fluorescence (at the 325nm place be for maximum excitation and at 392nm place emission maximum) strongly.

In the preferred embodiment of the invention, the lysine of peptide is terminal to be connected with nanodevice, makes the MCA group be released into cytosol when cleaved.The terminal useful synthetic handle (handle) for puting together of the lysine of peptide is because for example it can be connected base with difunctionality, such as the Acibenzolar radical reaction of Mal-PEG-Osu.Therefore, use the appearance of green fluorescence in the target cell that these methods produce that a kind of indication clearly is provided, promptly apoptosis begins (if cell is because of existing accumulative quantum dot to have redness, cell becomes orange from the color that merges so).

Be applied to extra fluorescent dye of the present invention and include but not limited to be reported to cis-parinaric acid Cell 75:241 (1993) such as () Hockenbery that DNA in the pair cell apoptosis sexual cell changes responsive acridine orange Development117:29 (1993) such as () Abrams and follows apoptotic lipid peroxidation sensitivity.Should notice that described peptide and fluorescent dye are only for illustrative.Expection effectively plays a part to can be applicable to the present invention as any peptides of the resultant aspartic acid specificity cysteine protease substrate of apoptosis.

VI. targeted constituent

As mentioned above, one other component of the present invention is that the nanodevice compositions can selectively targeted specific cell type (for example tumor cell).Although understanding mechanism not necessarily can implement the present invention and the present invention and be not limited to any specific mechanism of action, but in certain embodiments, nanodevice is by cell surface part targeted cells (for example tumprigenicity cell) and by the receptor-mediated endocytosis cell that is ingested.

The known lip-deep arbitrary portion of target cell (for example tumor cell) that is positioned at can be applied to the present invention.For example, being oriented to this class antibody partly makes compositions targeting of the present invention to the cell surface that contains this part.Perhaps, targeting moiety can be for being oriented to the part that is present in the receptor on the cell surface, or vice versa.In an embodiment preferred of the present invention, targeting moiety is a folacin receptor.In certain embodiments, targeting moiety is RGD peptide receptor (α for example _Vβ ₃Integrin).Similarly, vitamin also is used to make therapeutic agent targeting of the present invention to specific cell.

In certain embodiments of the invention, the targeting moiety composition that can also serve as a mark works.For example, tumor specific antigen is applied to the present invention, includes but not limited to carcinoembryonic antigen, prostate specific antigen, tryrosinase, ras, saliva Louis silk antigen (sialyly lewis antigen), erb, MAGE-1, MAGE-3, BAGE, MN, gp100, gp75, p97, protease 3, mucin, CD81, CID9, CD63; CD53, CD38, CO-029, CA125, GD2, GM2 and O-acetyl group GD3, M-TAA, M-fetus or M-urinary system.Perhaps, targeting moiety can for tumor suppressor protein, cytokine, chemotactic factor,, tumour-specific receptors ligand, receptor, apoptosis inducers or differentiation agent.

The tumor suppressor protein that expection is used for targeting includes but not limited to p16, p21, p27, p53, p73, Rb, Wilns tumor (WT-1), DCC, 1 type neurofibromatosis (NF-1), the sick tumor suppressor protein of Xi-Lin (VHL), Maspin, Brush-1, BRCA-1, BRCA-2, kinds of tumors Profilin (MTS), human melanoma gp95/p97 antigen, renal cell carcinoma-relevant G250 antigen, KS 1/4 pancarcinoma antigen, ovarian cancer antigen (CA125), prostate specific antigen, melanoma antigen gp75, CD9, CD63, CD53, CD37, R2, CD81, CO029, TI-1, L6 and SAS.Certainly, these only for typical tumor suppressor protein and expectation antigen of the present invention can with arbitrarily other for or become the activating agent coupling as the tumor suppressor producing protein as well known to those skilled in the art.

In the preferred embodiment of the invention, targeting is oriented to the factor by oncogene expression.They include but not limited to tyrosine kinase, both can be film-bonded, also can be for the kytoplasm form, such as the Src family member; Serine/threonine kinase is such as Mos; Somatomedin and receptor are such as platelet-derived somatomedin (PDDG); SMALL GTPases (G albumen) comprises the member among ras family, cyclin dependant protein kinase (cdk), the myc family member, comprises c-myc, N-myc and L-myc and bcl-2 and family member.

The present invention can targeting cytokine include but not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, ILA 1, IL-12, IL-13, IL-14, IL-15, TNF, GMCSF, beta-interferon and gamma interferon.Operable chemotactic factor includes but not limited to M1P1 α, M1P1 β and RANTES.

The present invention can targeting enzyme include but not limited to cytosine deaminase, hypoxanthine guanine phosphoribosyltransferase, galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase, glucocerebrosidase (glucocerbrosidase), sphingomyelinase, α-L-iduronidase, glucose-6-phosphate dehydrogenase (G6PD), HSV thymidine kinase and people's thymidine kinase.

The receptor and the associated ligands thereof that are applied in the context of the invention include but not limited to folacin receptor, adrenoreceptor, growth hormone receptor, lutropin receptor, estrogen receptor, EGF-R ELISA, fibroblast growth factor acceptor etc.

The hormone and the receptor thereof that are applied to targeting moiety of the present invention include but not limited to growth hormone, prolactin antagonist, galactagogin, lutropin, follicle stimulating hormone (foilicle-stimulatinghormone), chorionic-gonadotropin hormone, thyrotropin, leptin, thyroliberin (ACTH), angiotensin I, Angiotensin II, beta-endorphin, β-melanotropin (β-MSH), cholecystokinin, Endothelin I, galanin, Gastric inhibitory polypeptide (GIP), glucagon, insulin, amylopectin, lipotropin, GLP-1 (7-37) neurophysin and somatostatin.

In addition, the present invention's expection places the vitamin (fat-soluble and non--fatsoluble vitamin) of nanodevice targeted constituent can be used for the receptor that targeting has these vitamin, or otherwise is exactly the cell that absorbs these vitamin.Particularly preferably be fatsoluble vitamin in this respect, such as vitamin D and analog thereof, vitamin E, vitamin A etc. or water soluble vitamins, such as vitamin C etc.

In certain embodiments of the invention, the cancerous cell targeting group of any amount combines with dendritic macromole.The targeting dendritic macromole is puted together with the kernel tree dendritic macromolecules successively.Therefore, nanodevice of the present invention makes it have specificity (promptly say nothing of in conjunction with cancerous cell and do not combine with healthy cell) to target cancer cell.In addition, the multivalence dendritic macromole can be in conjunction with Polyethylene Glycol (PEG) or poly-ethyl  azoles quinoline (PEOX) chain so that the immunogenicity that helps to increase blood circulation time and reduce conjugate.

In the preferred embodiment of the invention, the targeting group with have short (for example directly coupling), in (it is basic for example to use little-molecule difunctionality to be connected, such as SPDP, sell by Pierce ChemicalCompany) or the dendritic macromole of long (for example the PEG difunctionality connects base, is sold by Shearwater Polymers) key put together.Because dendritic macromole has a large amount of functional groups, so more than one targeting groups can combine with each dendritic macromole.As a result of, between dendritic macromole and target cell, there is the multiple situation that combines.In these embodiments, dendritic macromole has high affinity by " collaborative combination " or multivalence interaction effect to its target cell.

Owing to the space reason, so part is more little, so can be in conjunction with the dendritic macromole surface many more.Recently, Wiener has reported the dendritic macromole with bonded folic acid and can be accumulated in the tumor cell surface of expressing high-affinity folacin receptor (hFR) and inside Invest.Radiol. such as (, 32:748 (1997)) Wiener especially.The hFR receptor obtains expressing or up regulation on epithelial tumor, comprises breast carcinoma.The control cells that lacks hFR does not show significantly accumulating of folate-deutero-dendritic macromole.Folic acid can combine by the carbodiimides coupling reaction with the PAMAM dendritic macromole in all generations.Folic acid is the targeting material standed for of good dendritic macromole, because it has less size and simply puts together operating procedure.

Bigger and still to belong to relatively little part be epidermal growth factor (EGF), promptly have the strand of 53 amino acid residues.Verified by connecting that PAMAM dendritic macromole that basic SPDP and EGF put together is bonded to human glioma cell's surface and by endocytosis (endocytosed), thus be accumulated in the lysosome Bio conjugate Chem. such as (, 7:7 (1996)) Casale.Because the EGF Rd is higher than on the normal cell 100 times reaching on the brain tumor cell, so EGF provides the useful targeting agent that is used for these tumor types.Because the EGF receptor is overexpression in mammary gland and colon cancer also, so EGF also can be as the targeting agent of these cells.Similarly, fibroblast growth factor acceptor (EGER) is also in conjunction with relatively little polypeptide class (FGF), and known many in them obtain expressing (particularly accompanying drawing 1,2 and 4) Int.J.Cancer61:170 (1995) such as () Penault-Llorca with high level in breast tumor cell line.

In the preferred embodiment of the invention, targeting moiety is antibody or antigen-binding fragments of antibodies (for example Fab unit).For example, the antigen of finding on many cancers (comprising mammary gland HER2 tumor) surface that is well studied is glycoprotein p185, and it only obtains expressing Oncogene 5:953 (1990) such as () Press in malignant cell.Even confirmed recombinant humanized anti--HER2 monoclonal antibody (rhuMabHER2) can suppress the breast cancer cell growth of overexpression HER2, and obtained evaluation (combining) (Proc.Am.Soc.Clin.Oncol. such as Pegram, 14:106 (1995)) with the conventional chemotherapy agent at the III clinical trial phase that is used for advanced tumor breast carcinoma.Park and Papahadjopoulos combine the Fab fragment of rhuMabHER2 with small unilamellar vesicle, can load chemotherapeutics doxorubicin (dox) and targeting tumor xenogeneic graft (Cancer Lett. such as Park to it then to overexpression HER2, Biochem. such as 118:153 (1997) and Kirpotin, 36:66 (1997)).These loaded dox's " immunoliposome " compare with corresponding not liposome or the free dox of the loading dox of targeting, show the general toxicity that cytotoxicity that tumor is increased and specific ionization dox reduce.

Antibody can be produced so that antigen or the immunogen (for example tumor, tissue or pathogen specific antigen) on can the various biological targets of targeting (for example pathogen, tumor cell, normal structure).This antibody-like includes but not limited to polyclone, monoclonal, chimeric, strand, Fab fragment and Fab expression library.

In certain preferred aspects, described antibody recognition tumour-specific epi-position (TAG-72 (Cancer Res.48:2214-2220 (1988) such as Kjeldsen for example; U.S. Pat 5,892,020; 5,892,019; And US5,512,443); People's cancer antigen (U.S. Pat 5,693,763; US5,545,530; And US5,808,005); TP1 and TP3 antigen (U.S. Pat 5,855,866) from the osteoma cell; Thomsen-Friedenreich (TF) antigen (U.S. Pat 5,110,911) from adenocarcinoma cell; From " the KC-4 antigen " of human prostate adenocarcinoma (U.S. Pat 4,708,930 and US 4,743,543); Human colorectal cancer antigen (U.S. Pat 4,921,789); CA125 antigen (U.S. Pat 4,921,790) from adenocarcinoma of bladder; From the DF3 antigen of human breast carcinoma (U.S. Pat 4,963,484 and US 5,053,489); HBT's antigen (U.S. Pat 4,939,240); Human melanoma p97 antigen (U.S. Pat 4,918,164); Cancer or orosomucoid O ALPHA1-Acid glycoprotein AGP-related antigen (CORA) (U.S. Pat 4,914,021); With people's prognosis of squamous cell lung cancer reaction but not with the human lung cancer antigen (U.S. Pat 4,892,935) of human small cell lung carcinoma reaction; T in the human breast carcinoma glycoprotein and Tn hapten (Carbohydr.Res.178:271-292 (1988) such as Springer); MSA breast cancer glycoprotein Br.J.Surg.75:811-817 (1988) such as () Tjandra of name; MFGM breast cancer antigen (Tumor Biol.10:12-22 (1989) such as Ishida); DU-PAN-2 cancer of pancreas antigen (Cancer Res.45:305-310 (1985) such as Lan); CA125 ovarian cancer antigen (Carbohydr.Res.178:29-47 (1988) such as Hanisch); YH206 LuCA (Hinoda etc. (1988) Cancer is (1988) J.42:653-658).Especially above-mentioned each list of references is incorporated herein by reference.

In other embodiment preferred, described antibody recognition specific pathogen (is for example invaded lung legionella (Legionella peomophilia), mycobacterium tuberculosis (Mycobacteriumtuberculosis), clostridium tetani (Clostridium tetani), hemophilus influenza (Hemophilus influenzae), Diplococcus gonorrhoeae (Neisseriagonorrhoeae), Treponoma palladium (Treponema pallidum), Bacillus anthracis (Bacillus anthracis), vibrio cholera (Vibrio cholerae), B. burgdorferi (Borrelia burgdorferi), corynebacterium diphtheriae (Cornebacteriumdiphtheria), staphylococcus aureus (Staphylococcus aureus), the human papillomavirus, the human immunodeficiency virus, german measles virus, poliovirus etc.).

Various operating procedure as known in the art can be used for producing polyclonal antibody.In order to produce antibody, can carry out immunity inoculation to various host animals by the peptide of injecting corresponding to required epi-position, include but not limited to rabbit, mice, rat, sheep, goat etc.In a preferred embodiment, make described peptide and immunogenic carrier put together (for example diphtheria toxoid, bovine serum albumin (BSA) or key hole  hemocyanin (KLH)).Various adjuvants are used for increasing immunne response according to the difference of host type, comprise Fu Shi (fully with incomplete) adjuvant; Mineral coagulant is such as aluminium hydroxide; Surfactant is such as LYSOLECITHIN SUNLECITHIN A; Pluronic Polyols; Polyanion; The peptide class; Oil emulsion; Key hole  hemocyanin; Dinitrophenol,DNP; With people's adjuvant of potentially useful, such as BCG (bacillus calmette-guerin vaccine) and Corynebacterium (Corynebacteriumparvum).

In order to prepare monoclonal antibody, can use provide produce antibody molecule by the continuous cell line in the culture technology (for example, referring to Harlow and Lane, Antibodies:ALaboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).They include but not limited to: the hybridoma technology (Kohler and Milstein, Nature 256:495-497 (1975)) of Kohler and Milstein research and development; And trioma technology; People B-quadroma technology (for example, referring to Immunol.Today 4:72 (1983) such as Kozbor) and (Cole etc. are at Monoclonal Antibodies and Cancer Therapy to produce the EBV-hybridoma technology of human monoclonal antibodies, Alan R.Liss, Inc., described in the pp.77-96 (1985)).

In extra embodiment of the present invention, can use state-of-the-art technology in germfree animal, to produce monoclonal antibody (for example, referring to PCT/US90/02545).According to the present invention, can end user's antibody and can end user's hybridoma Proc.Natl.Acad.Sci.U.S.A.80:2026-2030 (1983) such as () Cote or by transforming people's cell B in external use EBV virus (Cole etc. are at Monoclonal Antibodies and Cancer Therapy, Alan R.Liss is described in the pp.77-96 (1985)) obtain this antibody.

According to the present invention, for producing technology (U.S. Pat 4,946,778 that single-chain antibody is described; Be incorporated herein by reference) can be suitable for producing specific single-chain antibody.The present invention extra embodiment used to making up the technology that the Fab expression library describes Science 246:1275-1281 (1989) such as () Huse so as can be fast and easily evaluation have required specific monoclonal Fab fragment.

Can produce the antibody fragment of the idiotype (antigen binding domain) that contains antibody molecule by known technology.For example, this class fragment includes but not limited to: F (ab ') 2 fragments that can produce by the pepsin digestion of antibody molecule; Can be by the Fab ' fragment of reduction F (ab ') 2 segmental disulfide bond generations; Handle the Fab fragment that antibody molecule produces with passing through with papain and Reducing agent.

In the process of producing antibody, can by technology well known in the art carry out required antibody screening (for example radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoradiometric assay, GDP reaction, immunodiffusion are measured, original position immunoassay (for example, using aurosol, enzyme or radiosiotope), Western blotting, precipitation, gathering measure (example gel is assembled mensurations, hemagglutination mensuration etc.), complement in conjunction with measure, a-protein mensuration and immunoelectrophoresis mensuration etc.).

Dendritic macromole of the present invention system has the many advantages above liposome, such as its bigger stability, to the better control of size and polydispersity, and general lower toxicity and immunogenicity (for example, referring to Polymer Preprints 39:180 (1998) such as Duncan).Therefore, in certain embodiments of the invention, anti--HER2 antibody fragment and other targeting antibodies and dendritic macromole are puted together, as the targeting agent of nanodevice of the present invention.

In certain embodiments, with regard to cancer (for example breast carcinoma), use folic acid, EGF, FGF and antibody (or antibody fragment) targeted cells surface at the former MUC1 of tumor associated antigen, cMet receptor and CD56 (NCAM).The cell in case be ingested, nanodevice is with regard to the p53 in conjunction with (by the antibody of puting together) HER2, MUC1 or sudden change.

Difunctionality connects basic SPDP and SMCC and is connected base with long Mal-PEG-OSu and is used in particular for antibody-dendritic macromole and puts together.In addition, many tumor cells contain the surperficial agglutinin of binding oligosaccharide class, and wherein specific recognition is mainly owing to the latter's end carbon hydrate residue (Sharon and Lis, Science 246:227 (1989)).Suitable monosaccharide and non-glycosylated protein matter, the conjugate (Biochemie 70:1633 (1988) such as Monsigny) that provides specific ionization monosaccharide more to combine closely the tumor agglutinin such as combining of BSA.

Mannose group PAMAM dendritic macromole more closely reaches 400 times or more (Page and Roy, bioconjugates Chem., 8:714 (1997)) than monomer mannoside in conjunction with mannoside-binding lectin in conjunction with mannoside-binding lectin.Sialylated dendritic macromole and the combination in vitro and in vivo of other dendritic and the various sialates of inhibition-combination virus.By many monosaccharide residues (for example being used for the alpha-galactoside in conjunction with the cell of galactose) and dendritic macromole are puted together, produce the multivalence conjugate that the respective type tumor cell is had high affinity.Described attachment reaction be easy to by terminal amine be purchased α-galactosyl-phenyl isothiocyanate and react and carry out.The small size of carbohydrate allows high concentration to be present on the dendritic macromole surface.

Identifying and selecting the method very flexibly of suitable peptide targeting group is display technique of bacteriophage (for example, referring to Curr.Opin.Biotechol. such as Cortese, 6:73 (1995)), and it can use and be purchased test kit and carry out expediently.The phage display method produces the big and different combinatorial library of the peptide class that is attached to phage surface, and screening is used to combine closely at the peptide class of immobilization surface receptor.After combining closely, isolated viral construct and order-checking are so that identify peptide sequence.Use best peptide class to repeat this circulation as the starting point that is used for next peptide library.Finally identify suitable high-affinity peptide class and then it has been screened biocompatibility and target-specific.In this mode, can produce the peptide class that can put together dendritic macromole, have the multivalence conjugate that receptor in target cell (for example tumor cell receptor) or other required target is had high degree of specificity and affinity thereby produce.

Relate to being of above-mentioned targeting means " pre-targeting " means (for example, referring to Goodwin and Meares, Cancer (suppl.) 80:2675 (1997)).This tactful example comprises and beginning with tumour-specific monoclonal antibody and Succ-PEG-DSPE conjugate treatment patient.Use suitable biotinylation scavenger from blood flow, to remove remaining solubility conjugate.When the conjugate that is positioned tumor all keeps, import the biotinylation agent, interacting by powerful and specific biological element-Succ-PEG-DSPE thus is positioned tumor locus.Therefore, to make the radioactive dosage maximization with the approaching dosage of cancerous cell and its amount in the remainder health of cell that may impair one's health minimized.

Verified, if at first handle in conjunction with the Succ-PEG-DSPE molecule of polystyrene and introduce radiolabeled Succ-PEG-DSPE then with the biotinylation dendritic macromole, the Succ-PEG-DSPE molecule (Bio conjugate Chem. such as Wilbur, 9:813 (1998)) that reaches 4 labellings is arranged in each polystyrene-bonded Succ-PEG-DSPE so.Therefore, the biotinylation dendritic macromole can be used for method of the present invention, thereby works as the multivalence receptor that is used for the body radioactivity labelling, wherein the radioactive dosage in the each bonded antibody conjugates of gained is enlarged.In the preferred embodiment of the invention, one or more many-biotinylation assemblies on bunch shape dendritic macromole provide the multivalence target of radioactive label or boration Cancer Investigation 14:534 (1996) such as () Barth avidin or Succ-PEG-DSPE, thereby produce the radiological dose of the expansion that is used for tumor cell once more.

Dendritic macromole can also be used as scavenger, for example, is undertaken by making the dendritic macromole part biological elementization with multivalence galactose or mannose surface.Conjugate-scavenger complex can have extremely strong affinity to corresponding hepatocyte receptor then.

In other embodiment of the present invention, enhanced permeability and reservation (EPR) method is used for targeting.Enhanced permeability and reservation (EPR) effect is " passive " target tumor mode (referring to Duncan and Sat, Ann.Oncol., 9:39 (1998)) more.The EPR effect is concentrated for the macromole and the selectivity of granule in tumor microenvironment that are caused by the super permeability vascular system of tumor and extremely weak lymphatic vessel drain.Dendrimer composition of the present invention is used for this reason provides ideal polymer, and promptly they are harder relatively, and polydispersity is limited, and size and surface chemistry are controlled and have " load " the space, inside that can carry and discharge antitumor drug then.In fact, confirmed that PAMAM dendritic macromole-platinate can be accumulated in the solid tumor (the Pt level is higher than about 50 times of the Pt level that cisplatin obtains of using), and have cisplatin it is not had activity in vivo (Proc.Int ' l.Symp.Control.Rel.Bioact.Mater. such as Malik, Polymer Preprints39:180 (1998) such as 24:107 (1997) and Duncan) in the solid tumor models of effect.

Targeting moiety of the present invention can be discerned various other epi-positions on the biological target (for example on the pathogen).In certain embodiments, introduce molecular recognition elements so that identification, targeting or detect various Pathogenic organismss, include but not limited to: targeting HIV (Nature 333:426 (1988) such as Wies), influenza (Cell 56:725 (1989) such as White), chlamydiaceae (Chlamydia) (Infect.Imm.57:2378 (1989)), meningitis naphthalene Se Shi coccus (Neisseria meningitidis), Streptococcus suis (Streptococcus suis), Salmonella (Salmonella), parotitis, the sialic acid of newcastle and various viruses, described virus comprises reovirus, Sendai virus and myxovirus; 9-OAC sialic acid with targeting coronavirus, encephalomyelitis virus and rotavirus; Detect non--sialoglycoprotein of cytomegalovirus (Virology176:337 (1990)) and Measles virus (Virology 172:386 (1989)); The CD4 of targeting HIV (Nature 312:763 (1985) such as Khatzman), vasoactive intestinal peptide (J.of Neuroscience Research 18:102 (1987) such as Sacerdote) and peptide T (FEBS Letters 211:17 (1987) such as Ruff); The epidermal growth factor of targeting cowpox (Nature 318:663 (1985) such as Epstein); The rabic acetylcholinergic receptor of targeting (Science 215:182 (1982) such as Lentz); The Cd3 complement receptors of targeting Epstein-Barr virus (J.Biol.Chem.265:12293 (1990) such as Carel); The B-adrenergic receptor of targeting reovirus (Proc.Natl.Acad.Sci.82:1494 (1985) such as Co); The rhinoviral ICAM-1 of targeting (Nature 344:70 (1990) such as Marlin), N-CAM and myelin-associated glycoprotein MAb (Proc.Natl.Acad.Sci.85:7743 (1988) such as Shephey); The poliovirus receptor of targeting poliovirus (Cell 56:855 (1989) such as Mendelsohn); The fibroblast growth factor acceptor of targeting herpesvirus (Science 248:1410 (1990) such as Kaner); The few mannose of targeting bacillus coli (Escherichia coli); The ganglioside G.sub.M1 of targeting meningitis naphthalene Se Shi coccus (Neissetia meningitidis); With the antibody that detects extensive various pathogen (for example Diplococcus gonorrhoeae (Neissetiagonorrhoeae), Vibrio vulnificus (V.vulnificus), vibrio parahaemolytious (V.parahaemolyticus), vibrio cholera (V.cholerae) conciliate alginic acid vibrio (V.alginolyticus)).

In certain embodiments of the invention, targeting moiety is preferably nucleic acid (for example RNA or DNA).In certain embodiments, the targeting moiety of designing nucleic acid is so that by base pairing and specific nucleic acid (for example chromosomal DNA, mRNA or ribosomal RNA) hybridization.In other embodiments, nucleic acid binding partner or biological target.Identified in conjunction with following proteinic nucleic acid: the reverse transcriptase of HIV, Rev and Tat albumen (Gene 137 (1): 33-9 (1993) such as Tuerk); Growth factor of human nerve (Nuc.Acids Res.23 (16): 3198-205 (1995) such as Binkley); And VEGF (Biochem.83 (34): 10450-6 (1994) such as Jellinek).Preferably identify that by the SELEX operating procedure nucleic acid of binding partner is (for example, referring to U.S. Pat 5,475,096; US5,270,163; And US5,475,096; With at PCT publication number WO 97/38134, WO 98/33941 and WO 99/07724, all these documents are incorporated herein by reference), but many methods are as known in the art.

VII. synthesize and put together

It is synthetic and form and description that this class component and dendritic macromole are puted together to the invention provides each component in the nanodevice (promptly containing one or more each dendritic macromole in the mentioned component).

In the preferred embodiment of the invention, according to the synthetic preparation PAMAM dendritic macromole of typical branch (constituting macromole) by initiator nuclear.It comprises two one-step growths order, by the Michael addition of amino with two keys of acrylic acid methyl ester. (MA), uses ethylenediamine (EDA) to make terminal the carbomethoxy--(CO of gained subsequently ₂CH ₃) the amidatioon composition.

In the first step of this method, ammonia was reacted 48 hours down and in the inert nitrogen environment with MA (mol ratio: 1: 4.25) at 47 ℃.The gained chemical compound is called the 0th generation star-branching PAMAM three-ester.Next step comprises the PAMAM HN-3 (G=O) that makes three-ester and excessive EDA reaction and produce star-branching.In noble gas (nitrogen) environment and methanol, carry out this reaction and need finish in following 48 hours at 0 ℃.Repeat this Michael addition and amidatioon and produced for the 1st generation in proper order.

Synthetic first complete cycle of branch of PAMAM dendritic macromole has been finished in the preparation of this HN-3.Repeat (G=1-5) dendritic macromole that this reaction sequence causes synthetic higher generation (promptly be respectively ester-and amine-end capped molecule).For example, repeat this for the second time and produce the 1st generation that has six-ester and six-amine surface respectively in proper order.According to all subsequently 1-9 generation identical mode carry out identical reaction, thereby constituted the branch units layer of generation core-shell mechanism, this structure is as implied above to have accurate molecular weight and end group quantity.Can or make succinic anhydrides and the dendritic macromole (for example entirely for PAMAM, POPAM or POPAM-PAMAM hybridization dendritic macromole) that with amine is the surface react the dendritic macromole that generation is the surface with the carboxylate by the end capped PAMAM dendritic macromole of hydrolysis-ester.

Can be based on the synthetic various dendritic macromoles of the core texture that starts polymerization process.These core textures have been specified the structure key character of dendritic macromole, such as overall shape, density and surface functionality (Angew.Chem.Int.Ed.Engl. such as Tomalia, 29:5305 (1990)).The spherical dendritic macromole that derives from ammonia has trivalent initiator core, and EDA is four-valency initiator core.Recently, reported the clavate dendritic macromole based on poly-(aziridine) core of the linearity of different length, core is long more, then rod long more J.Am.Chem.Soc. such as (, 120:2678 (1998)) Yin.

In preferred embodiments, dendritic macromole of the present invention comprises protected core diamidogen.In particularly preferred embodiments, protected initiator core diamidogen is NH2-(CH2) _n-NHPG (n=1-10).In other embodiment preferred, the initiator core is selected from down group, includes but not limited to NH2-(CH2) _n-NH2 (n=1-10), NH2-((CH2) _nNH2) ₃(n=1-10) or not replace or replace 1,2-; 1,3-; Or 1,4-phenylene two-just-alkylamine wherein uses single protected diamidogen (NH2-(CH2) for example in per generation amide forming process _n-NHPG).In these means, protected diamidogen is the generation tree dendritic macromolecules on a large scale, does not characterize and analyze the nanostructured that produces difficulty and do not produce uneven may making.Only limit to an end by the reactivity with diamidogen, the chance of dimer/polymer formation and inner molecular reaction will be eliminated and need not to use a large amount of excessive diamidogen.Be easy to the terminal single protected intermediates of purification because protecting group for by conventional art, produce purification as crystallization and/or chromatograph suitable processing be provided.

Can make the protected intermediates deprotection in deprotection steps, and make the dendritic macromole in gained generation carry out ensuing chemical reaction repeatedly and need not purification, the present invention is not limited to specific protecting group.In fact; pay close attention to various protecting groups, include but not limited to tert-butoxy carbamate (N-t-Boc), allyloxy carbamate (N-Alloc), benzylamino formic acid esters (N-Cbz), 9-fluorenyl methyl carbamate (FMOC) or phthalimide (Phth).In the preferred embodiment of the invention, protecting group is benzylamino formic acid esters (N-Cbz).N-Cbz is ideal for the purpose of the present invention, and is cleaved by catalytic hydrogenation (Pd/C) under the condition because it is easy to " neutrality ", and need not to take strong acid or alkali condition to remove F-MOC.Protected monomer is applied to the high throughput production process especially, because can use the monomer of low amount, thereby has reduced production cost.

Can characterize the size and the uniformity of dendritic macromole by the analytical technology of any appropriate.They include but not limited to atomic force microscope inspection technique (AFM), electrospray ionization mass spectrum, MALDI-TOF mass spectrum, ¹³C nuclear magnetic resonance, NMR spectrophotography, high performance liquid chromatography (HPLC), size exclusion chromatography (SEC) (multiple angle laser light scattering, dual UV and refractive index detector have been installed), capillary electrophoresis and gel (get) electrophoresis.These analytical methods can be guaranteed the uniformity of dendritic macromole colony and be important aspect the quality control of dendritic macromole production, so that the application in finally using in vivo.The most important thing is, used dendritic macromole to carry out extensive work, when giving, do not demonstrated toxicity evidence (J.Biomed.Mater.Res. such as Roberts by intravenous, J.Magnetic Resonance Imaging such as 30:53 (1996) and Boume, 6:305 (1996)).

VIII. nanodevice antitumor efficacy and toxic evaluation

Can use nanodevice of the present invention to estimate the antitumor action of different therapeutic agents to cancerous cell line and primary cell culture.For example, in preferred embodiments, use tumor cell line model or the primary cultured cell (referring to for example embodiment 10-12) set up to test, perhaps, can use animal model to test (referring to for example embodiment 13) in vivo external.

A. vitro human tumor cell apoptosis-inducing is quantitative

In a typical embodiments of the present invention, nanodevice of the present invention is used for the apoptosis of the outer human tumor cells of detection bodies.The effect of therapeutic agent has been determined in apoptotic test in the pair cell.Can and should measure apoptotic many aspects.These aspects comprise those aforesaid aspects and following aspect, include but not limited to Phosphatidylserine (PS) is translocated in the plasma membrane mensuration of outer surface; The mensuration of dna fragmentationization; The detection of cell death related protein matter; And the active mensuration of aspartic acid specificity cysteine protease-3.

B. external toxicology

In certain embodiments of the invention, in order to obtain that the generality of the safety of specific nanodevice platform or this system components is seen clearly, carry out toxotest.Toxicology information can derive from many sources, includes but not limited to zooscopy in historical data base, testing in vitro and the body.

External in recent years toxicologic method has obtained popularizing, and this is because of due to the increase of the perception of the increase of zooperal selection needs and possible ethics, economy and scientific value.The in vitro toxicity test macro has many advantages, comprises that the effect of improvement, the cost of reduction and the variability between the experiment reduce.These systems also reduced animal use, eliminated chaotic general action (for example immunity) and controlled environmental condition.

Although any testing in vitro system can be used for the present invention, the most common means that is used for experiment in vitro is for using the cultured cells model.These systems comprise cell, primary cell or the cell transformed culture of fresh separated.As the external toxicologic cell culture of research because of the multiple culture of rapid screening but favourable, thereby on cell, subcellular fraction or molecular level, identify and estimate toxic action to be useful.External cell culture method disturbs by integrity, metabolic activity and the subcellular fraction of measuring film usually and demonstrates basic cytotoxicity.Film integrality indicator commonly used comprises that cell survival rate (cell counting), clone's expansion test, trypan blue are got rid of, endocellular enzyme discharges (for example lactic acid dehydrogenase), small ion (K ¹, Ca ²⁺) membrane permeability and micromolecule (for example ⁵¹Cr, succinate) born of the same parents in Ala accumulate.Subcellular fraction disturbs and comprises for example by MTT test monitoring mitochondria enzyme activity level, mensuration cell adenosine triphosphate (ATP) level, dimethyl diaminophenazine chloride absorption lysosome and quantitatively synthetic to gross protein.The metabolic activity indicator comprises glutathione content, lipid peroxidation (peroxiidation) and lactate/pyruvate ratio.

The C.MTT algoscopy

The MTT test is for obtaining the method for cell survival rate measured value fast, accurately and reliably.The MTT algoscopy is is at first researched and developed by Mosmann (Mosmann, J.Immunol.Meth., 65:55 (1983)).It is a kind of many laboratorys simple colorimetric method of being used to obtain the toxicity result (for example, referring to Arch.Toxicol. such as Kuhlmann, 72:536 (1998)).In brief, mitochondrion produces ATP so that provide enough energy for cell.In order to reach this purpose, mitochondrion metabolism pyruvate and produce acetyl-CoA.In mitochondrion, acetyl-CoA reacts the ATP that is produced subsequently with different enzyme in tricarboxylic acid cycle.The enzyme that is used in particular for MTT mensuration is a succinate dehydrogenase.MTT (bromination 3-(4,5-dimethylthiazole-2-yl)-2 diphenyl tetrazolium saltses) is a kind of yellow substrate that forms purple first  product by the succinate dehydrogenase cracking.Pigment alteration has been identified the change of mitochondrial function.Can not produce first  in the cell born of the same parents of not surviving, and the amount that directly produces thus is relevant with the amount of survivaling cell.The trap at 540nm place is used to measure the amount of first  product.

In vitro tests result and toxicity in vivo test are compared so that infer the animal condition (for example referring to embodiment 13) alive that obtains.Generally speaking, estimate the acute toxicity of single dose material.Any toxicity sign (body temperature increase, dyspnea, death etc.) of monitoring animal in 14 days.According to tradition, the acute toxicity standard is median lethal dose(LD 50) (LD ₅₀), it is the predicted dose when causing the death of half treatment colony.Measuring this dosage is undertaken by the dosage that makes test animal contact geometry under controlled condition learn series.Other test comprises the subacute toxicity test, and it measures the reaction that animal is no more than 14 days to the nanodevice of repeated doses.The subchronic toxicity test comprises repeated doses test 90 days.The chronic toxicity test is similar to inferior chronic test, but can continue 90-days more than the time limit.The body build-in test can also be carried out so that measure the toxicity of some organizational aspects.For example, in certain embodiments of the invention, measured tumor toxicity (being the effect of the present composition) (for example by detecting the change of tumor tissues size and/or growth) to the tumor tissues survival.

IX. gene therapy carrier

In specific embodiments of the present invention, dendrimer composition comprise be used for external, exsomatize or body in the transgenic that is delivered to target cell or tissue and is used to express.In this class embodiment, the dendritic macromole complex comprises the expression vector establishment body and does not contain actual protein, the various regulating elements that described expression vector establishment style produces in target cell as allogeneic dna sequence DNA that contains coding institute concern gene and the specified protein that helps being paid close attention to.

In certain embodiments, gene is labelling or the reporter gene that is used for the treatment of the therapeutic gene of cancer, substitutional defect gene or is used to select or monitor purpose.With regard to the gene therapy carrier, gene can be the allos fragment of DNA.Allogeneic dna sequence DNA can derive from more than one sources (being polygenes construct or fusion rotein).In addition, allogeneic dna sequence DNA can comprise adjusting sequence that derives from a kind of source and the gene that derives from separate sources.

Tissue-specificity promoter can be used for influencing transcribing of particular organization or cell so that reduce potential toxicity or unwanted effect to non-target tissue.For example, can be used for the gene expression of targeting prostate such as this class promoter of the special gonad kallikrein of PSA, probasin, prostate acid phosphatase or Prostato-(hK2).Similarly, promoter can be used for the gene expression of other tissue of targeting (for example insulin, elastin laminin amylase pdr-1, pdx-1 and glucokinase promoter targeting are to pancreas; Albumin PEPCK, HBV enhancer, alpha Fetoprotein apoC, α-1 antitrypsin, vitellogenin, NF-AB and transthyretin promoter targeting are to liver; Myosin H chain, creatine kinase, malnutrition gene, calpain p94, skeleton α-Ji Dongdanbai, fast troponin 1 promoter targeting are to skeletal muscle; Keratin promoter targeting skin; Sm22 α; SM-. α .-actin promoter targeting smooth muscle; CFTR; People's cytokeratin 18 (K18); Pulmonary surfactant protein A, B and Q CC-10; P1 promoter targeting lung tissue; Endothelin-1; E-selects albumen; Feng's von willebrand's factor; KDR/flk-1 targeting endothelium; Tryrosinase targeting melanocyte).

Nucleic acid can be cDNA or genomic DNA.Can the encode treatment albumen of any appropriate of nucleic acid.Preferred nucleic acid codes for tumor Profilin, cytokine, receptor, apoptosis inducers or differentiation agent.Nucleic acid can be antisensenucleic acids.In this class embodiment, antisensenucleic acids can be introduced the nanodevice of the present invention of expression vector environmental externality.

In preferred embodiments, nucleic acid coding tumor suppressor protein, cytokine, receptor or apoptosis inducers.Suitable tumor suppressor protein comprises BRCA1, BRCA2, C-CAM, p16, p211, p53, p73 or Rb.Suitable cytokine comprises GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, beta-interferon, gamma interferon or TNF.Suitable receptor comprises CFTR, EGFR, estrogen receptor, IL-2 receptor or VEGFR.Suitable apoptosis inducers comprises AdE1B, Bad, Bak, Bax, Bid, Bik, Bim, Harakiri or ICE-CED3 protease.

X. the method for conjoint therapy

The tumor cell of tolerance DNA damage agent has been represented the subject matter in the Clinical Oncology.Nanodevice of the present invention provides the mode of improving this difficult problem by effectively giving combination therapy approach.Yet, should note traditional conjoint therapy can with nanodevice coupling of the present invention.For example, in certain embodiments of the invention, can be before traditional remedies, unite the use nanodevice afterwards or with traditional remedies.

In order in conjoint therapy, to use method and composition killer cell of the present invention, cell growth inhibiting or transfer or blood vessel take place, and even the malignant phenotype who makes tumor cell reverses or reduces, and makes " target " cells contacting nanodevice compositions described herein and at least a other activating agent.Provide these compositionss with the combined amount of effectively killing and wounding or suppress cell tissue.This method can comprise makes cell contact described immunotherapeutic agent and the described activating agent or the factor simultaneously.Can comprise the single compositions or the pharmaceutical preparation of two kinds of activating agents by making cells contacting, or by making cell contact two kinds of different components simultaneously or preparation is realized this purpose, wherein a kind of compositions comprises: expression construct for example, and another kind of compositions comprises therapeutic agent.

Perhaps, the nanodevice tumor can be carried out before or after another kind of activating agent treatment in interval a few minutes to several weeks.Be applied to separately in the embodiment of cell at another kind of activating agent and immunotherapy, can guarantee that generally significant time bar can not interrupt between each Delivery time, make that activating agent and nanodevice still can the favourable synergy of pair cell performance.In this class situation, expection makes cell in about 12-24 hour, and two kinds of these the two kinds of forms (modality) that more preferably contacted with each other in about 6-12 hour, most preferably only about 12 hours time delay.Yet, in some cases, need be treatment significant prolongation time bar, wherein between corresponding administration, have several days (2-7) to the passing in several weeks (1-8).

In certain embodiments, use the administration of more than one immunotherapeutic compositions of the present invention or other activating agent.Can use various combinations, wherein dendritic macromole is " A ", and another kind of activating agent is " B ", the following is the typical case:

A/B/A，B/A/B，B/B/A，A/A/B，B/A/A，A/B/B，B/B/B/A，B/B/A/B，A/A/B/B，A/B/A/B，A/B/B/A，B/B/A/A，B/A/B/A，B/A/A/B，B/B/B/A，A/A/A/B，B/A/A/A，A/B/A/A，A/A/B/A，A/B/B/B，B/A/B/B，B/B/A/B。

Also expect other combination.In addition, in order to realize killer cell, two kinds of bioactive agent deliveries are delivered to cell effectively to kill and wound or to make the combined amount that cell loses vigor.

Can be used for including but not limited to produce the factor of DNA infringement with the other factors of the conjoint therapy of nanodevice of the present invention, such as. γ .-ray, X-ray and/or radiosiotope are to the targeted delivery of tumor cell.Also pay close attention to the DNA damage factor of other form, such as microwave and UV-irradiation.The dosage range of X-ray continues the time bar (3-4 week) of prolongation at dosage 50-200 roentgen's every day, to 2000-6000 roentgen's single dose.Radioisotopic dosage range can extensively change, and depends on the intensity of ray of isotopic half-life, emission and type and by the intake of tumprigenicity cell.Those skilled in the art are with reference to " Remington ' s Pharmaceutical Sciences " 15th Edition, and chapter 33, particularly the 624-652 page or leaf.On the dosage some changes the state of an illness that depends on the experimenter who is treated certainly.The people of responsible administration under any circumstance all can determine the suitable dose to individual subjects.In addition, for the human body administration, preparation should satisfy as the desired aseptic of FDA Office of Biologics standard, pyrogen, whole body safety and purity rubric.

In the preferred embodiment of the invention, nanodevice is used to make the treatment effectiveness maximization of active agent delivery to the patient's that suffers from cancer local delivery.Similarly, can make the nanodevice that comprises one or more functional groups (for example therapeutic agent, such as chemotherapeutics or radiotherapeutic agents) be oriented to experimenter's health specific be subjected to the infringement zone.Perhaps, in some cases, systemic delivery nanodevice (nanodevide) (dendritic macromole that for example comprises therapeutic agent, targeting agent and/or preparation) is suitable, for example, has wherein taken place extensively to shift or suspect and shift.

Except that merging nanodevice and chemotherapy and radiation, also pay close attention to and use traditional gene therapy.For example, the treatment of targeting p53 or p16 sudden change and nanodevice can provide the anticancer therapy of improvement.The present invention pays close attention to the co-therapy with other treatment related gene, includes but not limited to p21, Rb, APC, DCC, NF-I, NF-2, BCRA2, p16, FHIT, WT-I, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.

The suitable method that use is researched and developed for the gene delivery of the particular build body that is used for particular subject is used in the body and the treatment of exsomatizing.For example, with regard to viral vector, generally with 1 * 10 ⁴, 1 * 10 ⁵, 1 * 10 ⁶, 1 * 10 ⁷, 1 * 10 ⁸, 1 * 10 ⁹, 1 * 10 ¹⁰, 1 * 10 ¹¹Or 1 * 10 ¹²Infectious particles is delivered to the patient.Can infer similar data to liposome or other non-virus formulation by more corresponding ingestion efficiency.

The attractive local location that can therapeutic combination be delivered to the patient that is characterised in that of the present invention by medical apparatus.Be applicable to that medical apparatus of the present invention comprises the device that becomes known for the therapeutic agent local delivery.This class device includes but not limited to: conduit, such as injection catheter, balloon catheters, two balloon catheters, microporosity balloon catheters, passage balloon catheters, infusion catheter, perfusion catheter etc., for example, give activating agent to their coating therapeutic agents or by them; The needle-like injection device, such as hypodermic needle and needle-like injection catheter:; The needle-like injection device is such as jet injector; The support of coating, the support of bifurcated, blood vessel graft etc.; With the blood vessel-engaging device of coating, such as wire coil.

Typical devices is described in U.S. Pat 5,935,114; US5,908,413; US5,792,105; US5,693,014; US5,674,192; US5,876,445; US5,913,894; US5,868,719; US5,851,228; US5,843,089; US5,800,519; US5,800,508; US5,800,391; US5,354,308; US5,755,722; US5,733,303; US5,866,561; US5,857,998; US5,843,003; And US5,933,145; The full content of these documents is incorporated herein by reference.Be purchased and can be used for typical stent of the present invention comprise RADIUS (Scimed LifeSystems, Inc.), SYMPHONY (Boston Scientific Corporation), Wallstent (Schneider Inc.), PRECEDENT II (Boston ScientificCorporation) and NIR (Medinol Inc.).The target position that this class device is delivered to and/or implants by known technology.

XI. photodynamic therapy

In certain embodiments, treatment complex of the present invention comprises photodynamic compound and the targeting agent that the patient is given.In certain embodiments, make the targeting agent combine the certain hour time limit (for example about 1 minute-24 hours) with " target " cell then, thereby form target cell-targeting agent complex.In certain embodiments, shine the treatment complex (for example using red laser, electric filament lamp, X-ray or filterable daylight) that comprises targeting agent and photodynamic compound then.In certain embodiments, make light be oriented to jugular vein or some shows shallow blood vessel or lymphatic vessel.In certain embodiments, singlet oxygen and free radical diffuse to target cell (for example cancerous cell or pathogen) from photodynamic compound, thereby cause its destruction.

XII. pharmaceutical preparation

In certain embodiments of the invention, if pay close attention to clinical practice, prepare the ingredient of nanodevice so as the form pharmaceutical composition that is suitable for specifying application.Generally speaking, this requires preparation to be substantially free of pyrogen and other may be to the compositions of the deleterious impurity of human or animal.Yet, in certain embodiments of the invention, use in the approach described herein one or more to give straight chain dendritic macromole preparation.

In preferred embodiments, with nanodevice and suitable salt and buffer coupling so that with stable form delivering compositions, thereby can be absorbed by target cell.When nanodevice is imported the patient, can also use buffer.Aquo-composition comprises being scattered in the nanodevice of the cell effective dose in pharmaceutically acceptable carrier or the water-bearing media.This based composition is also referred to as inoculum.That term " pharmaceutically acceptable or pharmacology go up acceptable " intention does not produce when the animal or human is given is unfavorable, the molecule body and the compositions of anaphylaxis or other untoward reaction.Term used herein " pharmaceutically acceptable carrier " comprises arbitrarily and all solvent, disperse medium, coating material, antibacterial and antifungal, etc. blend absorption delayer etc.Except that with carrier of the present invention or inconsistent any typical media of cell or reagent, various their application in therapeutic combination.The active component of replenishing can also be mixed compositions.

In certain embodiments of the invention, active compound comprises traditional pharmaceutical preparation.The administration of these compositionss of the present invention is undertaken by approach commonly used arbitrarily, as long as target tissue can obtain by this approach.That this approach comprises is oral, nose, suck, rectum, vagina or part.Perhaps, administration can be undertaken by normal position, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.

Can also be by giving the active nano device in non-intestinal or intraperitoneal or the tumor.Preparation as the reactive compound of free alkali or the acceptable salt of pharmacology suitably being mixed with surfactant, such as the solution in the water of hydroxypropyl cellulose.Can also prepare the dispersion in glycerol, liquid macrogol and composition thereof and oil.Under the storage and service condition of routine, these preparations contain the antiseptic that prevents growth of microorganism.

In certain embodiments, the invention provides the compositions that comprises dendritic macromole, described dendritic macromole comprises targeting agent, therapeutic agent and preparation.In preferred embodiments, with dendritic macromole in vivo delivering therapeutic agents (for example methotrexate) to tumor cell (referring to for example embodiment 13, accompanying drawing 27).In certain embodiments, making therapeutic agent connect base by sensitivity to acid puts together with dendritic macromole.Therefore, in certain embodiments, (for example in the Inclusion) therapeutic agent discharges from dendritic macromole in target cell.Pay close attention in such born of the same parents and discharge (for example sensitivity to acid connects basic endosome (endosomal) destruction) so that provide to the present composition and the extra specificity of method.In preferred embodiments, dendritic macromole of the present invention (for example G5PAMAM dendritic macromole) contains 100-150 primary amine class (for example, referring to embodiment 13) from the teeth outwards.Therefore, the invention provides the dendritic macromole with a plurality of reaction site (for example 100-150) of puting together functional group, described functional group includes but not limited to treatment base, targeting agent, preparation and biological monitoring agent.

No matter whether dendrimer composition of the present invention comprises fluorescein (for example FITC) preparation, and the expection the compositions and methods of the invention are same effectively (for example, referring to embodiment 13).Therefore, each functional group that is present on the dendrimer composition can work independently with other functional group.Therefore, the invention provides the dendritic macromole of the combination that can comprise multiple targeting, treatment, imaging and biological monitoring functional group.

The present invention also provides molecule (for example treatment and imaging functional group) has been delivered to the inner very effective and specific method of target cell (for example cancerous cell).Therefore, in certain embodiments, the invention provides and comprise and maybe need molecule sent into cell so that the therapy that works (delivery of genetic material for example, siRNAs).

In certain embodiments, the pharmaceutical dosage form that is suitable for injecting application comprises aseptic aqueous solution or dispersion liquid and is used for preparing the sterilized powder of sterile injectable solution or dispersion liquid temporarily.Carrier can be for containing for example solvent or the disperse medium of water, ethanol, polyhydric alcohol (for example glycerol, propylene glycol and liquid macrogol etc.), its suitable mixture and vegetable oil.For example, can be by using coating material, such as lecithin, with regard to dispersion liquid by keeping required granular size and by using surfactant to keep suitable flowability.Can be with various antibacterial, antifungal, for example parabens, chlorobutanol, phenol, sorbic acid, thimerosal etc. produce the effect of prophylaxis of microbial.In many cases, preferably include isotonic agent, for example saccharide or sodium chloride.Can be by in compositions, using the reagent of delayed absorption, for example aluminum monostearate and gelatin prolong the absorption of Injectable composition.

Mix the suitable solvent that contains above-mentioned various other components by reactive compound and prepare sterile injectable solution, if desired, carry out aseptic filtration subsequently and prepare sterile injectable solution aequum.Generally speaking, by being mixed, various sterile active components contain alkaline disperse medium and from preparing dispersion liquid in above-mentioned those the aseptic vehicle of required other component.With regard to the sterilized powder that is used to prepare sterile injectable solution, preferred manufacturing procedure is vacuum drying and Freeze Drying Technique, and they can be produced the powder of active component and extra required arbitrarily component by above-mentioned aseptic filtration solution.

When preparation, give dendrimer composition in the mode compatible with such as upward effective this class consumption of treatment with dosage form.Be easy to give preparation such as Injectable solution, drug release capsules etc. with various dosage forms.For example, with regard to carry out parenterai administration with aqueous solution with regard to, if necessary, this solution is suitably cushioned, and liquid diluent at first makes with enough saline or glucose etc. and oozes.These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.For example, a kind of dosage can be dissolved in 1ml etc. oozes NaCl solution and joins in the 1000ml hypodermoclysis or in specified infusion site injection (for example, referring to " Remington ' sPharmaceutical Sciences " 15th Edition, 1035-1038 and 1570-1580 page or leaf).In certain embodiments of the invention, the amount of preparation of active particle or activating agent accounts for the 0.0001-1.0 milligram in the treatment mixture, or about 0.001-0.1 milligram or about 0.1-1.0, and even about 10 milligrams/dosage etc.Can give multiple dose.

The extra preparation that is suitable for other administering mode comprises vaginal suppository and vaginal suppository.Can also use rectum vaginal suppository or suppository.Suppository is the solid dosage forms of the common pastille of Different Weight and shape, and it is used to insert rectum, vagina or urethra.After the insertion, suppository softens, melts or is dissolved in chamber liquid.Generally with regard to suppository, conventional adhesive and carrier can comprise: for example poly alkylene glycol or triglyceride; This class suppository can be by containing 0.5%-10%, and the mixture of the active component of preferred 1%-2% forms.Vaginal suppository or vaginal suppository are generally spheric or avette and the about 5g of each deadweight.The vagina medicinal thing obtains with various physical form, for example cream, gel or liquid, and they have broken away from traditional suppository notion.In addition, suppository can be used for colon cancer.Nanodevice can also be mixed with the inhalant that is used for the treatment of pulmonary carcinoma etc.

XIII. the method for treatment or prophylaxis of cancer and pathogenicity disease

In specific embodiment of the present invention, provide the method and composition (for example, referring to embodiment 13) of treatment tumor in cancer therapy.Expect that therapy of the present invention can be used for the treatment of and identified specific marker or any cancer that can targeting.The cell proliferation sexual disorders or the cancer of various available the inventive method treatments include but not limited to: people's sarcoma and cancer include but not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, ewing's tumor, lymphangioendothelial sarcoma, synovioma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, wilms' tumor, cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, durosarcoma, melanoma, neuroblastoma, retinal neuroblastoma; Leukemia, acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia); Chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); With polycythemia vera, lymphoma (Hodgkin and non--Hodgkin), multiple myeloma, macroglobulinemia Waldenstron and heavy chain disease.

Various therapies of the present invention can be used for the treatment of has identified that specific marker maybe can be to specifying any pathogenicity disease of pathogen targeting.The example of the pathogen of available the inventive method treatment includes but not limited to: invade lung legionella, mycobacterium tuberculosis, clostridium tetani, hemophilus influenza, Diplococcus gonorrhoeae, Treponoma palladium, Bacillus anthracis, vibrio cholera, B. burgdorferi, corynebacterium diphtheriae, staphylococcus aureus, human papillomavirus, human immunodeficiency virus, german measles virus, poliovirus etc.

Experiment

It is for illustration and further explanation some preferred embodiment of the present invention and aspect that the following example is provided, but is not used for limiting scope of the present invention.

In the experiment disclosure hereinafter, use following abbreviation: g (gram); L or L (liter); μ g (microgram); μ l (microlitre); μ m (micron); μ M (micromole); μ mol (micromole); Mg (milligram); Ml (milliliter); Mm (millimeter); MM (mM); Mmol (mM); M (mole); Mol (mole); Ng (nanogram); Nm (nanometer); Nmol (nanomole); N (standard); Pmol (picomole); Aldrich (Sigma/Aldrich, Milwaukee, WI); Sigma (SigmaChemical Co., St.Louis, MO); Fisher Scientific (FisherScientific, Pittsburgh, PA); Millipore (Millipore, Billerica, MA); Mettler Toledo Mettler Toledo (Columbus, OH); Waters (WatersCorporation, Milford, Massachusetts); Wyatt Technology (WyattTechnology Corp., Santa Barbara, CA); TosoHaas (TosoHaas Corp., Montgomeryville, PA); Perkin Elmer (Perkin Elmer, Wellesley, MA); Beckman Coulter (Beckman Coulter Corp., Fullerton, CA); Phenomenex (Phenomenex, Torrance, CA); GiboBRL (GibcoBRL/LifeTechnologies, Gaithersburg, MD.); Pierce (Pierce ChemicalCompany, Rockford, IL.); Roche (F.Hoffmann-La Roche Ltd, Basel, Switzerland).

Embodiment 1

Material and method

At Center for Biologic Nanotechnology, the synthetic and sign G5PAMAM dendritic macromole of University ofMichigan.MeOH (HPLC level), acetic anhydride (99%), triethylamine (99.5%), DMSO (99.9%), Fluorescein isothiocyanate (98%), (+)-2,3-Epoxy-1-propanol (racemic form, 96%), DMF (99.8%), 1-(3-(dimethylamino)-propyl group)-3-ethyl carbodiimides HCl (EDC, 98%), citric acid (99.5%), Hydrazoic acid,sodium salt (99.99%), D ₂O, NaCl and the volumetric solution (0.1M HCl and 0.1M NaOH) that is used for constant-current titration are all available from Aldrich and as the use of standard.Methotrexate (99+%) and folic acid (98%) is from Sigma, Spectra/Por ^(PBS is pH=7.4) from FisherScientific for dialyzer (MWCO 3,500), Millipor Centricon ultrafiltration membrane YM-10 and phosphate buffered saline (PBS).

Potentiometric titration.Use Mettler Toledo MP230 pH Meter and MicroCombpH electrode under 23 ± 1 ℃ of room temperatures, to carry out manual titration.10mL 0.1M NaCl solution is joined in the 100mg PAMAM dendritic macromole of accurate weighing to prevent that amido from interacting.Use 0.1028N HCl to carry out titration and 0.1009N NaOH is used for back titration.Determine the quantity of primary amine class and tertiary amines according to the back titration data.

Gel permeation chromatography (GPC).2487 dual wavelength UV absorption detectors (Waters), Wyatt Dawn have been installed in use ^The Alliance Waters 2690 Separation Module of DSP laser spectrometer, Optilab DSP interferometry refractometer (Wyatt Technology) and use TosoHaas TSK-Gel ^Guard PHW 06762 (75 * 7.5mm, 12 μ m), G 2000 PW 05761 (300 * 7.5mm, 10 μ m), G 3000 PW 05762 (300 * 7.5mm, 10 μ m) and G 4000 PW (300 * 7.5mm, 17 μ m) post carry out the GPC experiment.With WatersTemperature Control Module column temperature is maintained 25 ± 0.1 ℃.Deng degree mobile phase is 0.1M citric acid and 0.025w% Hydrazoic acid,sodium salt, and pH 2.74, flow velocity 1ml/ minute.Sample concentration is 10mg/5ml, and volume injected is 100 μ L.Use Astra 4.7 softwares (Wyatt Technology) to measure the molecular weight and the molecular weight distribution of PAMAM dendritic macromole and conjugate thereof.

Nuclear magnetic resonance spectrometry: at D ₂Get among the O ¹H and ¹³C NMR spectrum and be used to provide integrated value is so that carry out structural analysis by Bruker AVANCE DRX 500 instrument.

The UV spectrophotography.Use Perkin Elmer UV/VIS spectrometer λ 20 and the UV spectrum of λ 20 software records in PBS.

Reversed phase high-performance liquid chromatography.Reversed phase ion pairing high performance liquid chromatography (RP-HPLC) system is by System GOLD ^TM126 solvent assemblies, Model 507 automatic samplers that 100 μ l rings have been installed and Model 166 UV detectors (Beckman Coulter) composition.To be used for separate analytes based on the HPLC post (250 * 4.6mm, 300 ) of Phenomenex Jupiter C5 silicon dioxide.(4 * 3mm) are contained in the upstream of HPLC post with two Phenomenex Widepore C5 guard columns in addition.The mobile phase that is used for eluting PAMAM dendritic macromole for 1ml/ minute flow velocity since 90: 10 water/acetonitriles (ACN), and after 30 minutes, reach 50: 50 linear gradient.Will the concentration in water and in ACN be the trifluoroacetic acid (TFA) of 0.14w% as counter ion counterionsl gegenions so that dendritic macromole-conjugate surface for hydrophobic.Described conjugate is dissolved in mobile phase (90: 10 water/ACN).Volume injected in each case is 50 μ l, sample concentration be about 1mg/ml and 210 242 or 280nm the sample of eluting is detected.Use Beckman ' s System GOLD ^TMNouveau software is analyzed.By using UV, HPLC, NMR and GPC that every kind of device and all intermediate are characterized.

The KB cell is available from ATCC (CLL17; Rockville, MD).Trypsin-EDTA, Dulbecco phosphoric acid-buffer saline (PBS), hyclone, cell culture antibiotic and RPMI culture medium are available from Gibco/BRL.All other reagent are all from Sigma.Synthetic and the sign of dendritic macromole-conjugate is reported to independent information.All dendritic macromole goods that use in this research are all synthetic at our center, and by free surface amino is carried out acetylation its surface neutralized.

Cell culture and processing.The KB cell maintained in the culture medium that contains 10% serum (for example, referring to Pharm.Res.19 such as Quintana, 1310 (2002)) that does not contain folic acid so as to provide with the similar born of the same parents that in human serum, find outside FA.Cell bed board in the flat board of 12-hole is carried out bed board so that carry out cell growth analysis so that absorb research in the flat board of 24-hole, and bed board is measured so that carry out XTT in the flat board of 96-hole.Wash cell and under 37 ℃, hatch specified time bar and concentration with the culture medium of the serum that contains dialysis that does not contain FA with dendritic macromole-drug conjugate.In addition the KB cell is maintained in the RPMI culture medium that contains 2 μ M FA so that obtain to express the cell of low FAR.

Flow cytometry and Laser Scanning Confocal Microscope art.Use the Beckman spectrofluorophotometer that the standard fluorescence of dendritic macromole solution is carried out quantitatively.For to the polymer of targeting picked-up carry out flow cytometric analysis, with the cell trypsinized and be suspended among the PBS that contains 0.1% bovine serum albumin (PBSB), and use Becton Dickinson FACScan analyzer to analyze.Measure the FL1-fluorescence of 10,000 cells and the survivaling cell fluorescence meansigma methods of gate is carried out quantitatively.Use Carl Ziess confocal microscope that the cell of bed board on the glass cover slide is carried out the confocal microscope analysis.Use argon laser, the suitable filter that is used for FITC is gathered fluorescence simultaneously and is differentiated interference contrast (DIC) image.

The Cytotoxic evaluation of dendritic macromole.By using two cinchoninic acid reagent (PIERCE) to detect that the gross protein of handling in the cell lysates is determined the cell growth and by using test kit to carry out XTT mensuration from Roche.

Embodiment 2

Synthesizing of dendritic macromole

Synthesize dendritic macromole (for example, referring to accompanying drawing 6) according to following method:

1.G5 carrier 7.G5-Ac ¹(82)-FITC-FA-MTX ^a

2.G5-Ac ³(82) 8.G5-Ac ³(82)-FITC-FA-OH

3.G5-Ac ³(82)-FITC 9.G5-Ac ³(82)-FITC-FA-OH-MTX ^e

4.G5-Ac ³(82)-FITC-OH 10.G5-Ac ²(82)-FA

5.G5-Ac ³(82)-FITC-OH-MTX ^e 11.G5-Ac ²(82)-FA-OH

6.G5-Ac ³(82)-FITC-FA 12.G5-Ac ²(82)-FA-OH-MTX ^e

(note: Ac ¹, Ac ², Ac ³Shown in subscript be used to distinguish not on the same group acetylization reaction).

1.G5 carrier.At Biologic Nanotechnology, the synthetic and sign PAMAM G5 dendritic macromole of University ofMichigan.The PAMAM dendritic macromole is made up of ethylenediamine (EDA) initiator core and 4 radial dendroid arms, and uses and to produce the reaction repeated sequence that each generation successively forms with a large amount of excessive EDA condensations (amidatioon) by the thorough Michael addition of acrylic acid methyl ester. (MA) and gained ester and synthesize.Successive reaction makes the quantity of surface amino groups double thus separately in theory, can make their activation so that functionalized.By analysis synthetic dendritic macromole and be 26 by the GPC determining molecular weight, 380g/mol, and be 110 with the quantitative measurement of primary amino radical by potentiometric titration.

2.G5-Ac ³(82)。Make 2.38696g (8.997*10 ^-5Mol) G5PAMAM dendritic macromole in 160ml abs.MeOH (measure MW=26 by GPC, 380g/mol is by quantity=110 of potentiometric determination primary amine class) and 679.1 μ l (7.198*10 ^-3Mol) acetic anhydride is having 1.254ml (8.997*10 ^-3Mol, 25% molar excess) triethylamine existence reaction down.After abundant dialysis and lyophilizing, obtain 2.51147g (93.4%) G5-Ac ³(82) product.Based on ¹The average (82) (Majoros, I.J., Keszler, B., Woehler, S., Bull, T., and Baker, J.R., Jr. (2003)) of acetyl group has been measured in H NMR calibration.

3.G5-Ac ³(82)-FITC。Make 1.16106g (3.884*10 ^-5Mol) the G5-Ac in 110mlabs.DMSO ³(82) PAMAM of partial acetylation (measures MW=29 by GPC, 880g/mol) with 0.08394g (90% is pure) (1.94*10 ^-4Mol) FITC reacts in nitrogen environment and spends the night.After abundant dialysis, lyophilizing, obtain the G5-Ac of 1.10781g (89.58%) ³(82)-the FITC product.Be further purified by membrane filtration.

4.G5-Ac ³(82)-FITC-OH。Make 0.20882g (6.51*10 ^-6Mol) G5-Ac ³(82)-FITC and 19.9 μ l (2.99*10 ^-4Mol) (+)-2,3-Epoxy-1-propanol (raceme) reacts in 150ml DI water.Each remaining primary amino radical is calculated two (+)-2,3-Epoxy-1-propanol molecules.With this reactant mixture vigorous stirring 3 hours at room temperature.After abundant dialysis 2 days and lyophilizing, G5-Ac ³(82)-productive rate of FITC-OH is 0.18666g (84.85%).

5.G5-Ac ³(82)-FITC-OH-MTX ^e。Make 0.02354g MTX (5.18*10 ^-5Mol) with 0.13269g (6.92*10 ^-4Mol) at room temperature reaction response 1 hour in 27ml DMF and 9ml DMSO, vigorous stirring simultaneously of EDC.This drips of solution is added to contains 0.09112g (2.72*10 ^-6Mol) G5-Ac ³(82)-the 150ml DI aqueous solution of FITC-OH in.With this reaction system vigorous stirring 3 days at room temperature.After abundant dialysis and lyophilizing, the molecule G5-Ac of targeting ³(82)-FITC-OH-MTX ^eProductive rate be 0.08268g (73.5%).

6.G5-Ac ³(82)-FITC-FA。Make 0.03756g (8.51*10 ^-5Mol) FA (MW=441.4g/mol) and 0.23394g (1.22*10 ^-3Mol) EDC (1-(3-(dimethylamino)-propyl group)-3-ethyl carbodiimides HCl; MW=191.71g/mol) in dry DMF of 27ml and the dry DMSO mixture of 9ml with under the nitrogen environment, reacted 1 hour.To be added drop-wise to 0.49597g (1.55*10 in this organic reaction mixture then ^-5Mol; MW=32,150g/mol) G5-Ac ³(82)-the DI aqueous solution (100ml) of FITC in.With this reactant mixture vigorous stirring 2 days.After dialysis and lyophilizing, G5-Ac ³(82)-FITC-FA weight is 0.5202g (98.1%).Be further purified by membrane filtration.

7.G5-Ac ¹(82)-FITC-FA-MTX ^a。Make 2.1763*10 ^-5MTX (MW=454.45g/mol) and the 3.0948*10 of mol (0.00989g) ^-4The EDC of mol (0.05933g) reacted 1 hour in dry DMF of 66ml and the dry DMSO mixture of 22ml with under the nitrogen environment.This organic reaction mixture is added drop-wise to 0.09254g (2.7051*10 ^-6Mol; Measure MW=34,710g/mol) G5-Ac by GPC ¹(82)-FITC-FA-NH ₂DI aqueous solution (260ml) in.With this solution vigorous stirring 2 days.After dialysis and lyophilizing, G5-Ac ¹(82)-FITC-FA-MTX ^aWeight is 0.09503g (96.5%).Before analysis, be further purified by membrane filtration.The trifunctional device is studied the cracked control compound of Chinese medicine as vitro cytotoxicity.

8.G5-Ac ³(82)-FITC-FA-OH。Make the 0.29597g (8.63*10 in 200ml DI water ^-6Mol) G5-Ac ³(82)-the PAMAM dendritic macromole conjugate of FITC-FA partial acetylation (measures MW=34 by GPC, 710g/mol) with 20.6 μ l (3.1*10 ^-4Mol, 25% molar excess) (+)-2,3-Epoxy-1-propanol (MW=74.08g/mol) reaction 3 hours.In fully dialysis, lyophilizing with repeatedly behind the membrane filtration, obtain 0.27787g (90.35%) G5-Ac of ization fully ³(82)-the FITC-FA-OH product.In in vitro study, do not observe non-specific uptake (referring to the part ii (24) of this research that is used to absorb research.

9.G5-Ac ³(82)-FITC-FA-OH-MTX ^e。Make 0.03848g (8.4674*10 ^-5Mol) MTX (MW=454.45g/mol) and 0.22547g (1.176*10 ^-3Mol) EDC reacted 1 hour in dry DMF of 54ml and the dry DMSO mixture of 18ml with under the nitrogen environment.This organic reaction mixture is added drop-wise to 0.16393g (4.6339*10 ^-6Mol; MW=36,820g/mol) G5-Ac ³(82)-the DI aqueous solution (240ml) of FITC-FA-OH in.With this solution vigorous stirring 3 days.In dialysis, repeatedly after membrane filtration and the lyophilizing, G5-Ac ³(82)-FITC-FA-MTX ^eWeight is 0.18205g (90.88%).

10.G5-Ac ²(82)-FA。Make FA two the step successive reactions in G5-Ac ²(82) combination.Make 0.03278g (7.426*10 ^-5Mol) the doubly excessive EDC 0.19979g (1.042*10 of FA and 14- ^-3Mol), then this FA-active ester drips of solution is added to the product G5-Ac of partial acetylation in 24ml DMF, the reaction of 8ml DMSO solvent mixture neutralization chamber relaxing the bowels with purgatives of warm nature ²(82) (0.40366g, 1.347*10 ^-5Mol) in the aqueous solution in 90ml water.After dialysis and lyophilizing, product weight is 0.41791g (96.7%).Quantity by UV spectrographic determination FA molecule.As extra sign, the GPC by the UV detector has been installed or do not observe free F A by agarose gel.

11.G5-Ac ²(82)-FA-OH。Make 0.21174g (6.60*10 ^-6Mol) list-sense dendroid device G5-Ac ²(82)-FA and 20.1 μ l (3.04*10 ^-4Mol) (+)-2,3-Epoxy-1-propanol reacted 3 hours in 154ml DI water and under the vigorous stirring.After dialysis and lyophilizing, have list-sense device (productive rate: 0.20302g, 91.05%) participation of the ization of hydroxyl and the conjugation reaction of methotrexate from the teeth outwards.

12.G5-Ac ²(82)-FA-OH-MTX ^e。In the solvent mixture of 7ml DMF and 9ml DMSO, make 0.02459g (5.41*10 ^-5Mol) MTX and 0.14315g (7.46*10 ^-4Mol) 1-(3-(dimethylamino)-propyl group)-3-ethyl carbodiimides (EDC) at room temperature with in the nitrogen environment reacted 1 hour.This reactant mixture of vigorous stirring.MTX-active ester drips of solution is added to the 0.09975g (2.95*10 that has hydroxyl from the teeth outwards in 150ml DI water ^-6Mol) in the list-sense dendroid device, and this reactant mixture at room temperature stirred 3 days.After dialysis and lyophilizing, test this difunctionality device G5-Ac with combination and biological substance ²(82)-FA-OH-MTX ^e(productive rate: 0.11544g, 93.9%).

Embodiment 3

Analyze the potentiometric titration curve of the terminal primary amino of G5PAPAM dendritic macromole

Carry out constant-current titration so that measure the quantity of uncle and uncle's amino.In theory, the G5PAMAM dendritic macromole has 128 primary amino radicals and 126 tertiary amine groups in its surface.Can measure these values by using mathematical model.Constant-current titration discloses on G5PAMAM dendritic macromole carrier surface and to have 110 primary amine classes (for example, referring to accompanying drawing 7, the titration curve of its expression by obtaining with 0.1M HCl volumetric solution direct titration and use 0.1M NaOH volumetric solution back titration).Use the average of the back titration data computation primary amino radical of using the acquisition of 0.1M NaOH volumetric solution.

The molecular weight of measuring each conjugate structure also is necessary, so that produce the fully multifunctional dendritic macromole of definition.To the GPC of multiple angle laser light scattering be installed and (for example be used for this purpose as the RI detector of concentration detector, referring to table 1, its representative have specified separately molecular weight and molecular weight distribution PAMAM dendritic macromole carrier and single-, two-and three-sense conjugate.Subscript numeral 2 and 3 (ex.-G5-Ac ²And G5-Ac ³) represent that they are two kinds of independently acetylization reactions).

Table 1

Embodiment 4

The dendritic macromole that is undertaken by gel permeation chromatography characterizes

Measure 26, the molecular weight of the G5 dendritic macromole of 380g/mol be lower than slightly theoretical value (28,826g/mol).These results have shown the deviation of ionic theory structure.Use the value (for example, referring to accompanying drawing 8) in the GPC data computation table 1 of a conjugate and calculate so that derive exact magnitude with described carrier-bound each functional group.Can be by from the conjugate that contains functional molecules

Deduction does not contain the conjugate of described functional molecules in the value

Value and calculate the average of each functional molecules divided by the molecular weight of functional molecular.G5-Ac can be provided ², G5-Ac ²(82)-FA-OH-MTX ^e, G5-Ac ³(82)-FITC-OH-MTX ^eAnd G5-Ac ²(82)-FITC-FA-OH-MTX ^eGPC elution profile (eluograms), wherein RI signal and laser light scattering signal are located overlapping (for example, referring to accompanying drawing 8) at 90 °.

Based on gpc analysis, the quantity that can measure FITC, FA, MTX and the (+)-2,3-Epoxy-1-propanol molecule puted together is (for example, referring to accompanying drawing 8:FITC:5.8, FA:5.7, MTX ^e: 5-6, OH:28-30).As a little higher than presumed value of measuring by GPC of the quantity of puting together molecule; This as a result the probability of maximum be that it has variable effect according to the difference of described device because of due to the effect of citric acid in the eluent.With these values and the value use in conjunction that obtains by NMR and UV spectrum analysis in accurate mensuration and the bonded quantity of respectively puting together molecule of dendritic macromole.

The theory and the defective chemical constitution (for example, referring to accompanying drawing 9) of G5PAMAM dendritic macromole are provided.Side reaction, such as bridging, and per generation produce the arm be less than theoretical predicted value and help to produce the different slightly structure of theoretical value representative with the G5PAMAM dendritic macromole.The defective chemical constitution of G5PAMAM dendritic macromole shows from per generation and loses arm, this may become problem, because they have destroyed the sphere of dendritic macromole, influenced the quantity of the bonded functional molecules of possibility thus and reduced the effect that each functional molecules may have in the cell of targeting.

Embodiment 5

The sign of dendritic macromole functional group

The acetylation of dendritic macromole.Acetyl turns to primary necessary step in the synthetic dendritic macromole.Partial acetylation is used to neutralize from the part on the dendritic macromole surface of intermolecular interaction in another reaction or the biosystem, prevents that thus non-specific interaction from taking place in building-up process and delivery process.Leave that the part of acetylizad surperficial amine can be in conjunction with functional group.Make remaining glycylization cause water solublity to increase (puting together the back) at FITC; make dendritic macromole more freely be dispersed in and have in the water-bearing media of targeting specific increase; thereby make it compare and more can be used as targeted delivery systems (Pharm.Res.19 such as Quintana, 1310 (2002)) with many typical media.

Thoroughly dialysis, lyophilizing and use PBS and DI water membrane filtration repeatedly are used to produce the G5-Ac of the partial acetylation of purification ²(82) and G5-Ac ³(82) PAMAM dendritic macromole (for example, referring to Macromolec such as Majoros 36,5526 (2003)).After at Fluorescein isothiocyanate (FITC) and (FITC-FA) puting together, according to (Wang etc. .Blood.15,3529 (2000)) in identical reaction sequence of finding the complete acetylation of dendritic macromole is studied for use in external picked-up.Thoroughly dialyse, lyophilizing and membrane filtration repeatedly, obtain complete acetylizad G5-Ac ¹(82)-FITC and G5-Ac ¹(82)-FITC-FA PAMAM.

When degree of acetylation increased, the diameter of dendritic macromole reduced, thereby had shown the anti-correlation (for example, referring to Macromolec such as Majoros 36,5526 (2003)) between degree of acetylation and the dendritic macromole diameter.Be used for protonated low quantity the primary amine class (with compare than low degree, under higher degree of acetylation) to cause structure to be subjected to the influence degree of electric charge-charge interaction lower, makes that thus structure is finer and close.Yet molecular weight has parallel dependency with degree of acetylation, and molecular weight increases with the increase of degree of acetylation.

Detect the fraction H of UV eluting at the 210nm place by monitoring ¹-NMR and HPLC further characterize PAMAM dendritic macromole (for example, respectively referring to accompanying drawing 10 (A) and (B)).The H of G5-Ac ¹-NMR spectrum is showed as follows: the peak that occurs at 4.71ppm is D ₂The representative of O, and be the representative of external standard two  alkane at the peak of 3.67ppm, and represent the methyl proton of acetamide at the peak of 1.89ppm.Peak 2.34ppm, 2.55ppm, 2.74ppm, 3.04ppm, 3.21ppm and 3.39ppm are the representative that is present in the proton in the acetylation dendritic macromole.

The structure of functional group.Provide have with the structure of FITC, FA and the MTX of asterisk labelled tree dendritic macromolecules bonded group (for example, referring to accompanying drawing 11, wherein on FA and MTX molecule labelling α-and γ-carboxyl).When the γ-carboxyl on the FA was used for puting together with dendritic macromole, FA had kept the strong affinity to its receptor, makes FA can keep it to play the ability of targeting agent effect.In addition, γ-carboxyl has the reactivity (for example, referring to Quintana, waiting Pharm.Res.19,1310 (2002)) that is higher than α-carboxyl at carbodiimides (carboiimide) in amboceptor and process amino coupled.

The H of functional group ¹-NMR.For the final quantity of measuring with the bonded all kinds of functional groups of dendritic macromole, must compare the H-NMR of functional group self and the H of the dendritic macromole puted together with functional group ¹-NMR.The H of functional group ¹-NMR (referring to, for example accompanying drawing 12): FITC H ¹-NMR-aromatic compound peak: 7.9ppm, 7.68ppm, 7.23ppm, 6.6ppm, 6.65ppm, 6.75ppm; FA H ¹-NMR-aromatic compound peak: 8.73ppm, 6.75ppm, 7.65ppm is at the D of 4.85ppm ₂O is at the CH of 3.3ppm ₃OD is at following aliphatic compound peak: 2.15ppm, 2.2ppm, 2.4ppm; With MTX H ¹-NMR-aromatic compound peak: 8.65ppm, 8.75ppm, 7.85ppm is at the D of 4.8ppm ₂O is at the CH of 3.35ppm ₃OD is at following aliphatic compound peak: 2.05ppm, 2.25ppm, 2.45ppm.

Embodiment 6

Puting together of functional group and acetylation dendritic macromole

Puting together of Fluorescein isothiocyanate and acetylation dendritic macromole.The G5-Ac of partial acetylation ³(82) the PAMAM dendritic macromole is used to put together Fluorescein isothiocyanate (FITC).Make the reaction of the dendritic macromole of partial acetylation and Fluorescein isothiocyanate, and, obtain G5-Ac in fully dialysis, lyophilizing with repeatedly behind the membrane filtration ³(82)-the FITC product.The thiourea key that forms is stable in the research process of device.

Puting together of folic acid and acetylizad list-sense dendritic macromole.The puting together of list-sense dendroid device of folic acid and partial acetylation carried out in condensation between the γ-carboxyl by folic acid and the primary amino radical of dendritic macromole.This reactant mixture is added drop-wise to contains G5-Ac ³(82)-the DI aqueous solution of FITC in and vigorous stirring 2 days (in nitrogen environment) so that FA and G5-Ac ³(82)-FITC puts together fully.Obviously the α carboxyl has participated in condensation reaction, but its reactivity is lower when comparing with the γ carboxyl.NMR also is used to confirm the quantity with the bonded FA molecule of dendritic macromole.With regard to the free FA in being present in sample, spike can occur in spectrum.Get free FA's ¹H NMR spectrum (referring to, for example accompanying drawing 12) and G5-Ac ³(82)-FITC-FA.G5-Ac ³(82)-FITC-FA spectrum in the broadening of aromatic compound proton peak show between FA and the dendritic macromole and have covalent bond.Based on the integrated value of aromatic compounds proton among methyl proton in the acetamido and the FA, the quantity of bonded FA molecule is calculated as 4.5.By the UV spectrographic method, use the quantity (4.8) of free FA concentration calibrating curve determining FA molecule.

MTX and acetylation be two-the puting together of sense dendritic macromole (passing through amido link).By G5-Ac ³(82)-the synthetic reference substance MTX trifunctional conjugate of FITC-FA.Anticarcinogen MTX commonly used and FA similarity structurally allow itself and G5-Ac ³(82)-FITC-FA is by being used to make FA and primary amino radical in conjunction with identical condensation reaction combination.Estimating from the mol ratio of reactant can be in conjunction with 5 drug molecule each dendritic macromole.Get the trifunctional device ¹H NMR spectrum.The broadening of aromatic compound proton peak shows between methotrexate and the dendritic macromole and has covalent bond.Based on the methyl proton in the acetamido and the integrated value of puting together aromatic compounds proton in the molecule, the quantity of bonded methotrexate molecule is calculated as 5.MTX puts together with acting on the comparison device that comparison MTX puts together by ester bond by amido link.Be connected with dendritic macromole by amido link with MTX and compare, methotrexate by ester bond in conjunction with being easier to the medicine cracking relatively and discharging into system.

Puting together of (+)-2,3-Epoxy-1-propanol and acetylation difunctionality dendritic macromole.Puting together of (+)-2,3-Epoxy-1-propanol and acetylation difunctionality device is important preorder step, so as by ester bond in conjunction with MTX with eliminate remaining NH ₂Thereby, avoid any unwanted non-specific targeting in the biosystem.(+)-2,3-Epoxy-1-propanol and G5-Ac ³(82)-and puting together of FITC-FA all remaining primary amino radicals are changed into alcohol radical, thus G5-Ac produced ³(82)-FITC-FA-OH.For the purpose that characterizes, MTX produces G5-Ac with puting together of the (+)-2,3-Epoxy-1-propanol dendroid device that contains FA or FITC ²-FA-OH-MTX ^E1* and G5-Ac ³-FITC-OH-MTX ^E2* (for example, referring to accompanying drawing 13 (A) and (B), be respectively the HPLC elution profile of each sample).

Embodiment 7

MTX and acetylation and (+)-2,3-Epoxy-1-propanol difunctionality dendritic macromole are puted together by ester bond

Characterize

Shown G5-Ac ²-FA-OH-MTX ^eH ¹-NMR (for example referring to, accompanying drawing 14).Put together the proton of aromatic compound in the device peak representative can't with the H at free FA and MTX ¹The peak of the aromatic compound of finding among-the NMR is distinguished mutually.Aromatic protons is bimodal appearance at 6.59ppm, 7.53ppm, and is unimodal appearance at 8.37ppm.H with free FA and free MTX ¹-NMR and the H that puts together device ¹-NMR compares and shows that the aromatic compounds district is almost similarly overlapping, can not measure the position of aromatic compounds proton thus.The quantity of the binding molecule of FA and MTX also influences the distribution at peak.Represented solvent D at the peak that 4.70ppm occurs ₂O, the peak that occurs at 3.67ppm is the representative of external standard two  alkane, and is the representative of the methyl proton of acetamido at the peak that 1.89ppm occurs.Peak 2.31ppm, 2.52ppm, 2.71ppm and 3.26ppm are the representative of dendritic macromole proton.

Embodiment 8

The UV spectral characterization of dendritic macromole

The MTX of test by ester bond puts together and puts together comparison in the improvement aspect the cracking by amido link and dendritic macromole.By using EDC chemical bond MTX.Shown G5-Ac-FITC-FA-OH-MTX at 305nm ^eHPLC elution profile (for example, referring to accompanying drawing 15).Can with the merging UV spectrum of the FA that dissociates, MTX and FITC and G5-Ac (82), single-, two-and the UV spectrum of three-sense dendritic macromole compare (for example, respectively referring to the accompanying drawing in the accompanying drawing 16 and 17).UV spectrum provides accurately determines the peak at the FA of 281nm and 349nm, for MTX successively at 258nm, 304nm and 374nm, and to FITC at 493nm.The visible difference of FA, FITC and MTX peak (for example, referring to accompanying drawing 16) depends on puting together of each molecule and dendritic macromole.The UV spectrum of the material by relatively dissociant and dendritic macromole-put together is used for determining function in conjunction with dendritic macromole to the sign of every kind of device.

Embodiment 9

The cellular uptake of dendritic macromole

Use the fluorescence of the standard solution of fluorescent spectrophotometer assay conjugate G5-FI, G5-FITC-FA and G5-FITC-FA-MTX.Observed linear relationship between dendritic macromole concentration and the fluorescence at 10-1000nM.The fluorescence of the 100nM solution of G5-FITC, G5-FITC-FA and G5-FITC-FA-MTX is respectively 0.57,0.23 and 0.11 fluorescence spectrophotometry analytical unit.Difference on these fluorescence can be indicated because of having FA and MTX quencher on the dendritic macromole.

Measure the cellular uptake of dendritic macromole in the KB cell of expressing high cell surface FA receptor (FAR).The dendritic macromole that FA-puts together combines with cell in the dose dependent mode, wherein the combination rate to G5-FITC-FA and G5-FITC-FA-MTX is 50% under 10-15nM, do not contrast dendritic macromole G5-FITC (for example, referring to accompanying drawing 18A) and in the KB cell, detect.When the fluorescence that will obtain during, G5-FITC-FA and G5-FITC-FA-MTX have been obtained identical binding curve (for example, referring to accompanying drawing 18B) to observed quench correction in the dendritic macromole standard solution.The binding kinetics analysis of G5-FITC-FA-MTX (100nM) is presented in 30 minutes obtained Bmax, this result is to similar to the result of free folic acid (folate) combination rate report.

Tested the effect that free FA absorbs dendritic macromole in the KB cell of expressing high and low FAR.For G5-FITC-FA and G5-FITC-FA-MTX, conjugate and bonded 30% (for example, referring to accompanying drawing 19, the left group) that is combined into the cell of expressing high FAR of expressing the KB cell that hangs down FAR.The dendritic macromole (30nM) that 50 μ M FA have blocked targeting fully express low-and the cell of height-FAR in picked-up (for example, referring to accompanying drawing 19, left group).Estimate combining and endocytosis of dendritic macromole and KB cell by the Laser Scanning Confocal Microscope art.The KB cell was hatched 24 hours with the specified dendritic macromole of 250nM and get confocal images.The conjugate that contains target molecule FA was taken in KB cell (for example, referring to accompanying drawing 20) in 24 hours.Compare with the cell of handling with the contrast conjugate, the cell of contact G5-FITC-FA-MTX has shown by the inductive cytotoxicity of medicine-conjugate than inadhesion and gathering.

Embodiment 10

The dendritic macromole cell growth inhibiting that functional group puts together

Because being combined in, conjugate and KB cell reach maximal oxygen value 2002.Pharmaceutical Research 19:1310-1316. such as () Quintana in 1 hour, begin in the culture medium that does not contain medicine, to hatch 5 days subsequently by cell is hatched the effect of testing the G5-FI-FA-MTX cell growth in 1 hour with conjugate.Under this class condition, conjugate can not show any growth inhibited effect in the KB cell.When cell is hatched 4 hours with dendritic macromole,, there is about 10% cell growth moderate reduction as by the XTT test determination.Carried out cytotoxic assay in minimum 24 hours by hatching with dendritic macromole thus, this time bar is to demonstrate the preincubate time bar of inducing significant cytotoxicity.

The time-histories and the dose-dependent inhibition of cell growth.Above-mentioned research is verified, if completely lost FA in the culture medium, so at the external inductive cytotoxicity of MTX-that detects (for example, referring to Sobrero; Bertino, Int.J.Cell Cloning 4,51 (1986)).The effect of trifunctional dendritic macromole cell growth in the cell that test is hatched in the insufficient culture medium of FA-.Cell was handled 1-4 days and by estimating that cell protein content measures cell proliferation with 300nM conjugate (being equivalent to 1500nM MTX) or 1500nM free MTX.Conjugate or free MTX with variable concentrations are handled 2 days (conjugate concentration is appointed as the MTX equivalent, 5 MTX are wherein arranged in each dendritic macromole) with cell.The KB cell of expressing high and low FAR was hatched 1 hour flushing and by flow cytometric analysis mensuration cell fluorescence (for example, referring to accompanying drawing 21, left group) under 37 ℃ with the dendritic macromole of 30nM.Prevented cell combination and picked-up polymer conjugate (for example, referring to accompanying drawing 21, left group) in 30 minutes fully with the free FA preincubate of 50 μ M.

Also the XTT experimental test by XTT being changed into first  based on active wire plastochondria through living cells to the inhibition (for example, referring to JImmunol Methods such as Roehm 142,257 (1991)) of the inductive cell growth of conjugate.G5-FITC or G5-FITC-FA pair cell in the time of 1,2 or 3 day does not have the growth inhibited effect, and G5-FITC-FA-MTX and free MTX show time-dependent cytotoxic (for example, referring to accompanying drawing 22).Therefore, G5-FITC-FA-MTX and free MTX with time-and dose dependent mode cell growth inhibiting, and the contrast dendritic macromole can not cell growth inhibiting (for example, referring to accompanying drawing 21 and 22).

Embodiment 11

The inductive cytotoxicity of methotrexate does not take place in folic acid rescue cell

When the inductive growth inhibited of free MTX is higher than the inductive growth inhibited of MTX among the G5-FITC-FA-MTX that is being lower than 1 μ M of molar concentrations such as use (for example, referring to accompanying drawing 21), whether the FA part among the test G5-FITC-FA-MTX can save cell, and the inductive cytotoxicity of MTX-does not take place.The MTX and the FA of molar concentrations such as the G5-FITC-FA-MTX goods contain measure the free MTX of similar concentration and the effect that free FA cell growth suppresses.Under free FA that waits molar concentration and MTX, FA has reversed the inductive cell growth inhibited of MTX (for example, referring to accompanying drawing 23).With 150 or 500nM MTX at the free FA of molar concentrations such as being with or without the KB cell was handled 24 hours.Also use 30 and 100nM G5-FI-FA-MTX (be equivalent to 150 and 500nM MTX) parallel processing cell.The flushing cell is so that remove medicine and hatched 6 days with fresh culture medium again, and measures total cell protein matter.The 150nM FA that exists has almost completely reversed the growth retardation that is caused by 150nM MTX.In addition, similar by the inductive cytotoxicity of G5-FITC-FA-MTX (for example, referring to accompanying drawing 23, filled squares symbol) and equimolar FA and MTX combination (for example, referring to accompanying drawing 23, solid circles symbol).

When the inductive cytotoxicity of MTX-does not take place for free FA blocking-up dendritic macromole picked-up and rescue cell, tested the antiproliferative effect of cell and excessive FA preincubate to G5-FITC-FA-MTX.Make the KB cell not or the conjugate of contact variable concentrations or free MTX are arranged in the presence of the 50 μ M FA 24 hours.Remove and hatch culture medium, flushing cell and hatched again 5 days with fresh culture in the presence of the medicine not having, and carry out the XTT test (for example, referring to accompanying drawing 24,, ■, representative is with the cell of MTX processing; △, ▲, the cell that representative is handled with G5-FITC-FA-MTX).Excessive free FA has not only blocked growth inhibited, and the cell that increases growth is higher than cellular control unit 20% (for example, referring to accompanying drawing 24).

Embodiment 12

The stability of dendritic macromole

The stability of testing tree dendritic macromolecules in cell culture medium is so that check whether MTX discharges before entering cell from dendritic macromole.G5-FITC-FA-MTX with cell culture medium incubation 1,2,4 and 24 hours, and is used 10, and the ultrafiltration apparatus that 000-MW ends filters the incubation culture medium.Test trapped substance and filtrate are to the effect of KB cell growth.G5-FITC-FA-MTX was hatched 24 hours with the culture medium of 2 μ M concentration.The membrane filtration incubation culture medium of ending by 10K-MW.Trapped substance (being adjusted to the pre-filtering volume) and filtrate and KB cell (under the 200nM conjugate, as measuring according to the pre-filtering sample concentration) hatched 2 days together and carry out the XTT test.To available from having obtained similar result with filtrate to the trapped substance of dendritic macromole precincubation 1,2 and 4 hours culture medium.In 24 hours incubation time bar processes, trapped substance is Cytotoxic, and filtrate does not show any cytotoxicity (for example, referring to accompanying drawing 25), shows that free MTX can't discharge from conjugate.MTX slowly discharges after 24 hours, reaches the maximum that 40-50% discharges in 1 week.

The antiproliferative effect of MTX-conjugate is compared with the conjugate that lacks FA or FITC molecule.The KB cell was hatched 24 hours and removed and hatch culture medium with the conjugate (the effective MTX concentration of=150nM) of 30nM.Hatched again 5 days in flushing cell and the fresh culture in the presence of not having medicine, and carry out the XTT test.Lacking dendritic macromole that the MTX-of FA puts together can not inducing cytotoxic, and the dendritic macromole of targeting is not or inducing cytotoxic in the presence of the dye molecule FITC (for example, referring to accompanying drawing 26) arranged.

Embodiment 13

Dendritic macromole is the application in the target tumor in vivo

Compositions of the present invention (for example multifunctional dendritic macromole) and method are used for measuring the therapeutic response of animal model for cancer (for example HEP's cancer).

Material and reagent.All reagent are all available from merchandise resources.Folic acid, penicillin/streptomycin, hyclone, IV Collagen Type VI enzyme, TX100, two-benzimide (benzimide), FITC, methotrexate, hydrogen peroxide, acetic anhydride, ethylenediamine, methanol, dimethyl formamide and DMSO available from Sigma-Aldrich (St.Louis, MO).Trypsin-EDTA, Dulbecco ' s PBS and RPMI 1640 (being with or without folic acid) from Invitrogen (Gaithersburg, MD)." soluble " solution and hionic fluorophor from Packard Bioscience (DownersGrove, IL).OCT embedding culture medium is from Electron Microscopy Sciences (FortWashington, PA), the 2-methybutane is from Fisher Scientific (Pittsburgh, PA), and 6-carboxyl tetramethylrhodamin (6-TAMRA) and Prolong are from MolecularProbes, Inc. (Eugene, OR).Acetic anhydride (the CH of tritium-labelling ₃CO) ₂O[ ³H] (100mCi, 3.7GBq) available from ICN Biomedicals (Irvine, CA).Methotrexate for inj from Bedford Laboratories (Bedford, OH).Folic acid is dissolved in saline, is adjusted to pH 7.0 and carries out aseptic filtration so that injection with 1N NaOH.

Synthetic and the sign of PAMAM dendritic macromole conjugate.Synthesize the G5PAMAM dendritic macromole and purification (for example, referring to Macromolecules such as Majoros 36,5529 (2003)) from low molar mass pollutant and high molecular weight dimer or oligomer.By the size exclusion chromatography, use multiple angle laser light scattering, UV and refractive index detector that the mumber average molar mass of dendritic macromole is determined as 26,530g/mol.Use constant-current titration and molal weight that the par of the surperficial primary amine groups in the dendritic macromole is determined as 110.The polydispersity index that ideal monodispersity sample is defined as weight-average molar mass and the ratio of mumber average molar mass equals 1.0.The polydispersity index of G5 dendritic macromole is calculated as 1.032, and showing around meansigma methods has extremely narrow distribution and turns out to be highly purified G5 dendritic macromole.Use acetic anhydride with the surface amine acetylation of G5PAMAM dendritic macromole so that reduce the non-specific binding of dendritic macromole.The ratio of selecting acetic anhydride and dendritic macromole is so that obtain the different acetylation levels of 50-80 and 100 primary amine classes.Behind purification, acetylizad dendritic macromole and preparation (for example FITC or 6-TAMRA) are puted together so that detect and imaging.Make (for example dyestuff-the put together) dendritic macromole of imaging-put together and the Acibenzolar reaction of targeting agent (for example folic acid) then, and pass through ¹H nuclear magnetic resonance, NMR (NMR) is analyzed the product of this reaction purification, so that measure the quantity of the targeting agent (for example folate molecule) of puting together.Put together therapeutic agent (for example methotrexate) (for example, referring to Pharm Res such as Quintana 19,1310 (2002)) by ester bond subsequently.

Use the acetic anhydride (Ac-of tritiate ³H) by G5-(Ac) ₅₀-(FA) ₆Or G5-(Ac) ₅₀The chemical compound of synthesizing radioactive labelling is (for example, referring to J Control Release 65,133 (2000) such as Malik; Pharm Res such as Nigavekar 21,476 (2004); Bioconjug Chem such as Wilbur 9,813 (1998)).Make conjugate, the G5-of tritiate ³H-FA and G5- ³The complete acetylation of H.G5-NHCOC- ³H and G5-FA-NHCOC- ³The activity specific of H conjugate is respectively 10.27 and 38.63mCi/g.The gross activity of remaining free tritium＜0.3%.

Use PAGE, ¹H NMR, ¹³The quality of C NMR and mass spectrography test PAMAM dendritic macromole conjugate.The purity and the uniformity that capillary electrophoresis are used to confirm end-product.

The conjugate of folic acid-targeting contains following molecule: G5-(Ac) especially ₈₂-(FITC) ₅-(FA) ₅, G5-(Ac) ₈₂-(6-TAMRA) ₃-(FA) ₄, G5-(Ac) ₈₂-(FITC) ₅-(FA) ₅-MTX ₅And G5-(Ac) ₅₀-(Ac-3H) ₅₄-(FA) ₆, use acronym G5-FI-FA, G5-6T-FA, G5-FI-FA-MTX and G5-3H-FA to identify them respectively.The reference substance of non-targeting contains following molecule: G5-(Ac) ₈₂-(FITC) ₅, G5-(Ac) ₈₂-(6-TAMRA) ₃, G5-(Ac) ₈₂-(FITC) ₅-MTX ₅And G5-(Ac) ₅₀-(Ac-3H) ₅₄, use acronym G5-FI, G5-6T, G5-FI-MTX and G5-3H to identify them respectively.

Receptor and tumor model.The female nude mice of immunodeficiency type 6-to 8-week athymism in age [Sim:(NCr) nu/nu fisol] available from Simonsen Laboratories, Inc. (Gilroy, CA).The serious associative form immunodeficiency (SCID of 5-to 6-week Fox Chase in age; CB-17/1crCrl-scidBR) female mice is available from Charles RiverLaboratories (Wilmington, MA), and according to regulations and the federal guideline of University ' s Committeeon the Use and Care of Animals, it is indoor to comprise that Principles of Laboratory Animal Care makes them reside in the zoopery that does not contain special pathogen of University of Michigan Medical Center.Animal can arbitrarily absorb Laboratory Autoclavable RodentDiet 5010 (PMI Nutrition International, St.Louis, MO).The injection tumor cell before 3 weeks, with food change into folic acid-shortage meals (TestDiet, Richmond, IN).For gather urine and feces, make animal reside in metabolizable rodent cage (Nalgene, Rochester, NY) in.

Tumor cell line.The KB human cell line of overexpression folacin receptor (for example, referring to JCell Sci 106 such as Turek, 423 (1993)) available from American Type TissueCollection (Manassas, VA) and with them maintain external 37 ℃, 5%CO2 down and folate-shortage replenished penicillin (among the RPMI 1640 of 100 units/mL), streptomycin (100 μ g/mL) and 10% heat-inactivated fetal bovine serum.Before injecting mice, use trypsin-EDTA solution collecting cell, wash and be suspended among the PBS again.The 30-gauge needle with cell suspension (in 0.2mL 5 * 10 ⁶Individual cell) percutaneous injects the flank of every mice down.In biodistribution research, make 2 weeks of tumor growth, up to reaching～0.9cm ³Volume.The formula that select to calculate gross tumor volume at be ellipsoidal normal volume, V=4/3 π (1/2 long * 1/2 wide * 1/2 degree of depth) wherein.Widely equal the degree of depth and k equals 3 owing to infer, so the formula that uses is V=1/2 * length * wide ²The 4th angel brings into use the medicine of passing of conjugate injection carrying out targeting after implanting the KB cell.

The bio distribution of tritiate dendritic macromole and drainage.Contain 174 μ g G5-NHCOC-by lateral tail vein to the animal injection ³H (1.8ACi) or 200 μ g G5-FA-NHCOC- ³The 0.5mL PBS solution of H (7.7 μ Ci).With etc. the dendritic macromole of modification of molar concentration send the tritium-labeled conjugate of two kinds of tritiates.When injection back 5 minutes, 2 hours, 1 day, 4 days and 7 days, animal is implemented euthanasia, and get the sample of tumor, the heart, lung, liver, spleen, pancreas, kidney and brain.The 3rd group of mice is at injection 200 μ g G5- ³Accepted the free bolus injection of 80 μ g before H-FA5 minute.The free folic acid of this 181nmol concentration produces～150 μ mol/L concentration in blood, and by comparison, the dendritic macromole (G5-of radiolabeled targeting ³H-FA) in blood, produce～5 μ mol/L concentration, and based on the blood volume of 1.2mL 20g mice.When injection back 5 minutes, 1 day and 4 days, animal is implemented euthanasia 5, and gather tissue as mentioned above.Take a blood sample at each time point by cardiac puncture.Every group comprises 3-5 mice.When 2,4,8 and 12 hours and 1,2,3 and 4 day, gather urine and fecal specimens.

As Nigavekar etc. at the radioactivity of preparation described in the Pharm Res 21,476 (2004) tissue sample.(LS 6500, Beckman Coulter, Fullerton, CA) mensuration tritium level to use liquid scintillation counter.Adjust the radioactive values of measuring so that add up the efficient of instrument and be used to derive radioactivity (1 μ Ci=2.22 * 10 ⁶Dpm)/sample.Calibrate these values with tissue weight then and the specificity radioactivity of conjugate is reported to the percentage ratio (%ID/g) of injected dose.To be reported to the percentage ratio (%ID) of injected dose by the radioactivity (dendritic macromole) of urine and defecate.

The bio distribution of fluorescence dendritic macromole conjugate.The 0.5mL saline that contains 0.2mg G5-6T or G5-6T-FA conjugate by lateral tail vein to injected in mice.Back 15 hours of injection with when reaching 4 days, animal is implemented euthanasia and gets tumor sample and freezing immediately so that section and imaging.Use the unicellular suspension that separates from tumor to implement flow cytometry.The chopping tumor is by 70 μ m nylon sieves (Becton Dickinson, Franklin Lakes, NJ) filtration cell suspension and wash with PBS.Use EPICS XL flow cytometer (Coulter, Miami, FL) analytic sample.As what measure, for analyzing only gate living cells by the dyeing of third ingot of iodate in advance.Data are reported to the passage fluorescence meansigma methods of cell mass.

In order to carry out the confocal microscope imaging, cut open from tissue, be embedded among the OCT and in the dry ice bath, be chilled in 2-methyl-butane.Section (15 μ m) on cryostat is melting fixing on the microscope slide and being stored under-80 ℃, till dyeing.After dyeing, microscope slide is fixed in 4% paraformaldehyde, with phosphate buffer (0.1mol/L; PH 7.2) flushing, and be fixed among the Prolong.The Zeiss 510 metal laser scanning confocal microscopes that x40 Plan-Apo 1.2 numerical apertures (water immersion) object lens that have corrector loop have been installed in use obtain image.Confocal images is recorded as 512 * 512 * 48 pixels, and its scale that has is each pixel 0.45 * 0.45 * 0.37 μ m.With each image cube cut into 48 sections by optical instrument, and provide section by nucleus and Cytoplasm cutting.

Sending of the nano-particle therapeutic agent of targeting.Implanted the back the 4th day from tumor, weekly 2 times through the SCID of the subcutaneous KB of giving xenograft mice by tail vein injection accept targeting or non-targeting the conjugate that contains methotrexate, do not contain methotrexate conjugate, the methotrexate or the saline of product in contrast dissociate.Send chemical compound with 0.2mL volume saline/20g mice.The single dose of the methotrexate of at every turn sending equals 0.33mg/kg.Also tested the free methotrexate of 1.67 and 3.33mg/kg of higher dosage.With calculate based on the quantity that is present in the methotrexate in the nanoparticle etc. the methotrexate of molar concentration send conjugate.With etc. the dendritic macromole of molar concentration send the conjugate that does not contain methotrexate.In initial test, every group only has 6 groups of mices of 5 mices to accept to reach 15 injections.In test subsequently, according to the difference of its survival condition, mice accepts to reach 28 injections.Monitoring mice body weight in whole experiment is as the indication of adverse effect.When each test stops, carry out many organ-tissues pathology and measure, and because of toxic action or tumor load mice is implemented euthanasia at every turn.Analysis is from the tissue of lung, the heart, liver, pancreas, spleen, kidney and tumor.In addition, isolated cell from tumor is used the conjugate dyeing of the fluorescein-labelling of targeting, and uses the existence of flow cytometer test folacin receptor.

Statistical method.The meansigma methods of calculating data, SD and SE.Use Student ' s-Newman-Keuls ' to check difference between experimental group and the matched group, and Ps＜0.05 is thought of as has significance.

The bio distribution of tritiate dendritic macromole.Realize check tritiate G5- ³The bio distribution of H-FA and elimination are so that test the ability of folacin receptor-positive human KB tumor xenogeneic graft that its targeting sets up in the immunodeficiency nude mice.In The time limit of experiment, mice is kept folic acid-shortage meals, so that the cyclical level of folic acid is reduced to bottom line (for example, referring to J Nucl Med29 such as Mathias, 1579 (1998)).Before experiment is near the human serum level, in mice serum, obtain free folate level (referring to Anal Biochem such as Belz 265,157 (1998); AnalBiochem such as Nelson 325,41 (2004)).Time points (5 minutes-7 days) different after intravenous gives conjugate are estimated mice.2 groups of mices are accepted the tritiate G5-of non-targeting ³The tritiate G5-of H dendritic macromole reference substance or targeting ³H-FA conjugate (accompanying drawing 27A and B).Conjugate is removed from blood fast by kidney in the 1st day after injection, wherein G5- ³1.8%ID/g (accompanying drawing 27A) when H drops to 24 hours from the tissue of the 23.4%ID/g 5 minutes the time.G5- ³0.2%ID/g (accompanying drawing 27B) when the blood drug level of H-FA drops to 24 hours from the 29.1%ID/g 5 minutes the time.In several organs, in lung, tissue distribution has shown and G5- ³1.6%ID/g and G5-when H drops to 24 hours from the 9.7%ID/g 5 minutes the time ³The similar trend of blood drug level of 1.7%ID/g when H-FA drops to 24 hours from the 9.6%ID/g 5 minutes the time.Because many vasculars of lung, can reflect blood drug level in the conjugate level of each time point determining.The heart, pancreas and spleen have been observed similar removing pattern.Known these organs are not expressed folacin receptor (folatereceptor) and do not demonstrate significant difference between the dendritic macromoles of non-targeting and targeting.G5- ³H and G5- ³The concentration of H-FA in brain is all low at all time point places, points out described polymer conjugate not by blood brain barrier (accompanying drawing 27A and B).Although kidney is the main removing organ of these dendritic macromoles, known it also be expressed in high-caliber folacin receptor on its tubule.The G5-of non-targeting ³The level of H in kidney descends fast and maintained appropriate level (accompanying drawing 27A) in ensuing several day.On the contrary, G5- ³The horizontal pro-of H-FA increases a little in 24 hours and most probable reason is to exist on the renal tubules folacin receptor.After this in ensuing several days, descend, because chemical compound is removed (accompanying drawing 27B) by kidney.

G5- ³H and G5- ³H-FA is main by the kidney rapid drainage in back 24 hours of injection.Two kinds of chemical compounds are drained and are increased the kidney reservation (accompanying drawing 27A and B) in full accord that seems with conjugate.Although the conjugate of targeting and non-targeting also appears in the feces, its amount is still extremely low.Whether actual the drainage in feces because of the urine in the feces pollutes is difficult to measure any material.The G5-of targeting ³Accumulation clearance rate in the H-FA pro-4 days is lower than G5- ³The accumulation clearance rate of H, this reflects G5- ³H-FA keeps in the tissue of expressing folacin receptor.Known liver and the high-caliber folacin receptor of KB tumor cells expression.In these tissues, the G5-of non-targeting ³The concentration of H with dendritic macromole from blood removing and descend fast; Concentration maintains low-level (accompanying drawing 27A) in the natural law of remaining research organization.On the contrary, in liver and tumor, the G5-of targeting ³H-FA content pro-increased (accompanying drawing 27B) in 4 days.When radioactivity conjugate blood drug level is low, this situation takes place, to have pointed out on the contrary with the situation of holding back dendritic macromole by vascular system merely, the specificity at the dendritic macromole of Concentraton gradient in these tissues absorbs.

At injection G5- ³Accept further to have found in the mice group of the free folic acid of 181nmol the specificity (accompanying drawing 27C) of targeted delivery of drugs before the H-FA., in not having the tumor tissues of draining the dendritic macromole ability, observed and blocked the relevant radioactivity of folacin receptor with free folic acid and significantly weaken (accompanying drawing 27C) in the time of back 4 days in injection.Tumor concentration difference between the polymer conjugate of this prompting targeting and non-targeting is because of due to the specificity of these molecules by the folacin receptor of overexpression in tumor absorb.Before the conjugate of injection targeting, significantly do not change bio distribution in all other tissues by sending free folic acid.

The targeting and the absorption of fluorescence dendritic macromole conjugate.In order further to confirm and the in-house dendritic macromole nanoparticle of positioning tumor, use the dendritic macromole of puting together with 6-TAMRA.Behind the G5-6T conjugate of the G5-6T-FA of intravenous injection targeting and non-targeting, from tumor sample, obtain confocal microscope image (accompanying drawing 28) 15 hours the time.Compare (accompanying drawing 28A) with the dendritic macromole that uses non-targeting, use the tumor tissues of the dendritic macromole G5-6T-FA of the dyestuff of targeting-put together to demonstrate a large amount of fluorecytes (accompanying drawing 28B).To separate fluidic cell quantitative analysis from the unicellular suspension of identical tumor shown from the tumor cell of the mice of accepting G5-6T-FA than high channel fluorescence (accompanying drawing 28C).

Confocal microscope shows that also conjugate is present in the tumor, combines with many tumor cells and by its absorption (accompanying drawing 28D).Get the optical superposition part from the top through the centre the microscope slide to organizing of basilar part.Tumor cell middle slice provided herein shows the fluorescence from conjugate 6T in whole cytosol, wherein cell and nucleus sharpness of border visible (accompanying drawing 28D).

The toxicity of dendritic macromole conjugate.In the research time limit, observe all mices dehydrations, can not diet or the sign that changes of drinking-water, weakness or level of activation.When reaching 99 days, do not observe the acute or chronic serious toxicity that exists, whether contain methotrexate with the dendritic macromole conjugate and have nothing to do.In whole experiment, monitor body weight and do not observe and lose weight; In fact, the weight increase of animal.When each time point, liver,spleen,kidney, lung and the heart are totally checked and histopathological examination.When histopathological examination, do not observe paramophia.Toxicity in vivo not to be noted in any animal group of injection behind the dendritic macromole.

The medicine of targeting is delivered to tumor cell by folacin receptor.The conjugate of test various dose to the effect of SCID CB-17 mice with subcutaneous people KB xenograft and with etc. the free methotrexate of dosage and higher dosage compare.Mice is kept folic acid-3 weeks of shortage meals, after this inject the KB tumor cell so that obtain near in the human serum and the folic acid cyclical level (referring to J Nucl Med 29,1579 (1998) such as Mathias) that the folacin receptor decrement is regulated on the prophylaxis of tumours xenograft.Have in giving every group under the side flank percutaneous of 6 groups of SCID mices of 5 mices and be injected at 5 * 10 among the 0.2mL PBS ⁶The KB cell suspension.The highest accumulated dose of used G5-FI-FA-MTX therapeutic agent equals 55.0mg/kg and is equivalent to total cumulative dose (accompanying drawing 29) of the free methotrexate of 5.0mg/kg.The therapeutic dose of conjugate three kinds of cumulative dose with the free methotrexate that is equivalent to be accumulated in based on the mice survival rate 10-15 time 33.3,21.7 and 5.0mg/kg in injecting are compared.With saline and the conjugate (G5-FI-FA) that does not contain methotrexate as reference substance.

Monitoring mice body weight in whole experiment, as the indication of adverse effect, and body weight changes acute and chronic toxicity under the free methotrexate that is presented at the highest and inferior the highest cumulative dose and equals 33.3 and 21.7mg/kg respectively.Although the free drug of two kinds of dosage all influences tumor growth, they become (accompanying drawing 29) of lethal by the end of 32-36 days of this test the time.Remaining experimental group has very weight fluctuations uniformly, when comparing with the matched group that uses saline or do not contain the conjugate of methotrexate, does not show toxicity.With regard to the free methotrexate of used the highest cumulative dose, the histopathological analysis of liver is disclosed the hepatic injury in late period, inflammatory cell is assembled and peripylephlebitis disease.On the contrary, be equivalent to total cumulative dose of treatment conjugate of the free methotrexate of 5.0mg/kg and the equal avirulence of free methotrexate (accompanying drawing 29) under the same dose.Importantly, the free methotrexate (21.7mg/kg, 13 injections) of conjugate therapeutic dose and time maximum dose level that equals the lowest dose level of used free methotrexate has the effectiveness that is equal to.And the free drug under this concentration does not act on (accompanying drawing 29) to tumor growth, and the conjugate (G5-FI-FA) that does not contain methotrexate does not have therapeutical effect (accompanying drawing 29) when comparing with the matched group of pump pickle.Liver microscope slide from the mice of accepting conjugate (G5-FI-FA-MTX) shows all lymphocytes of accidental portal vein, indicates non-inflammation and the unicellular necrosis that is different from the control animals of pump pickle.

In second 99-days process of the test, growing to such an extent that have significance,statistical (P＜0.05) aspect slow without the tumor of FITC treatment than the tumor of those G5-FI-MTX conjugates, dissociate methotrexate or brine treatment with non-targeting with G5-FI-FA-MTX or G5-FA-MTX conjugate.The dose,equivalent that uses the conjugate of two kinds of targeting to be delivered to the methotrexate of survival mice is higher than the dosage of free methotrexate, because all accept mice death (accompanying drawing 30) when the 66th day of this test of free methotrexate.The mice survival rate of accepting in the group of G5-FI-FA-MTX or G5-FA-MTX conjugate shows that the tumor growth of the 4cm3 volume during based on terminal point can be delayed at least 30 days in (accompanying drawing 30).This numerical tabular is understood the antitumor effectiveness of conjugate, because it has been simulated clinical endpoint and need observe mice in whole progression of disease process.In addition, when the 39th day of this test, in 1 mice of using the G5-FA-MTX conjugate to treat, obtained to cure fully.Tumor in this mice is subsequently 20 days, and is untouchable when the 60th day of this test.When this test stops, accept 3 (8 merely hit) survivors that have of G5-FA-MTX, and accept G5-FI-FA-MTX 2 survivors (8 merely hit) are arranged.In the group of accepting free methotrexate or do not have the mice survival in other matched group arbitrarily.Therefore, in certain embodiments, the invention provides the compositions that comprises dendritic macromole, described dendritic macromole comprises targeting agent, therapeutic agent and preparation.In preferred embodiments, dendritic macromole is used in vivo therapeutic agent delivery (for example methotrexate) to tumor cell in the targeting specific mode.

Effective dose avirulence based on body weight change of carrying out and histopathological examination discovery conjugate.When two kinds of tests stopped, histopathological examination did not demonstrate the cardiac toxicity sign and myopathy does not take place.In these animals, do not observe the acute tubular necrosis in the kidney.The analysis of tumor microscope slide is shown that the tumor of surviving in the animal of matched group and pump pickle only has slight necrosis, and the treatment conjugate is compared with isodose free methotrexate and is caused severe to arrive the significance necrosis in tumor.When this test stops, compare the evaluation tumor cell with the KB cell because of the long-term folic acid of mice-possible up regulation of shortage meals folacin receptor in tumor.Fluidic cell quantitative analysis with the tumor cell after the conjugate dyeing of the fluorescein-labelling of targeting is disclosed cell kept the folacin receptor positive, but be lower than original KB cell line 2-5 doubly.

Embodiment 14

Synthesizing of PAMAM-dendritic macromole-RGD4C peptide conjugate

Drug targeting is crucial for effective cancer chemotherapy.Targeted delivery can improve the chemotherapy effect and make normal structure avoid the toxic side effects of these violent medicines.The angiogenesis inhibitor therapy forms (for example, referring to Los and Voest, Semin.Oncol., 2001,28,93) by inhibition of endothelial cell proliferation, migration and differentiation prevention new vessels.The evaluation that can distinguish the molecular marker of the new blood capillary that forms by its ripe counter pair provide with the cytotoxic agent targeted delivery to the mode of tumor vascular system (for example, referring to Br.J.Cancer such as Baillie, 1995,72,257; Ruoslahti, Nat.Rev.Cancer, 2002,2,83; Science such as Arap, 1998,279,377).α _Vβ ₃Integrin has specific a kind of labelling most in these unique tag.

Only found α on the surface, endotheliocyte chamber in the blood vessel generating process _Vβ ₃Integrin.This labelling can be by being limited to the spatial targeting agent identification of blood vessel (referring to Science such as for example Brooks, 1994,264,569 in the blood vessel generating process; Cleaver and Melton, Nat.Med .2003,9,661).Identified α by phage display research _Vβ ₃The high affinity of selective ligands Arg-Gly-Asp (RGD) (Nat.Biotech. such as Pasqualini, 1997,15,542).The RGD of peptide of dual cyclisation (RGD4C contains 2 disulfide bond by 4 cysteine residues) and conformation constraint is easier in conjunction with α than peptide class with single disulphide bridges or linear peptides class _Vβ ₃To synthetic be used for gene delivery (for example, referring to J.Gene.Med. such as Kunath, 2003,5,588-599), cancer target (for example, referring to J.Controlled Release such as Mitra, 2005,102,191) and imaging applications (for example, referring to J.Nucl.Med. such as Chen, 2004, the concern of polymer 45,1776) (poymer)-RGD conjugate is increasing.

In certain embodiments, the invention provides synthetic with fluorescently-labeled the 5th generation RGD4C that puts together of dendritic macromole.In addition, the invention provides the binding characteristic and the cellular uptake of these conjugates.

The dendritic macromole of amine termination combines with cell in the non-specific mode owing to positive surface charge according to reports.In order to improve the targeting effect and to reduce non-specific interaction, use acetic anhydride (75% * molar excess) having triethylamine (for example to carry out the part surface modification as the G5 dendritic macromole that in the presence of the alkali amine is stopped, referring to Macromolecules such as Majoros, 2003,36,5526.4).Initial passing through to the PBS buffer and then to water dialysis purification conjugate.The application of the acetic anhydride of 75 molar excess breaks away from some amidos so that further modify and prevent the problem that the dissolubility because of gathering, intermolecular interaction and reduction produces.

Can use ¹The degree of acetylation and the purity of H NMR spectrographic method monitoring acetylation G5 dendritic macromole (G5-Ac).Detect conjugate in order to pass through flow cytometry or confocal microscope art, but can use detector probe (for example fluorescent probe).For example, can be with Alexa Fluor488 (AF) as fluorescent labeling.The dendritic macromole of partial acetylation and the described scheme of Alexafluor-NHS ester such as manufacturer of 5 molar excess are reacted, and obtain fluorescently-labeled conjugate (G5-Ac-AF).By gel filtration with this conjugate of purification of dialysing subsequently.By ¹HNMR and UV-vis spectrographic method according to the described scheme of manufacturer (Molecular Probes), with the quantity survey of dye molecule are in each dendritic macromole～3.

The RGD peptide (RGD4C) that is used for certain embodiments of the invention has with the high-affinity specificity in conjunction with α _Vβ ₃The RGD sequence of conformation constraint.Assorted dimerization α _Vβ ₃Fracture place of RGD binding site between two subunits in the integrin.For the bound fraction of the peptide that keeps in touch target site, with ε-Aca (acyl group caproic acid) at interval base be used to make peptide and dendritic macromole to put together.Thus, the protonated NH of RGD-4C peptide ₂Terminal is not requisite for biological activity.Therefore, in certain embodiments, give NH with acetyl group ₂End-capped (for example, referring to Mol.Cancer Therap. such as deGroot, 2002,1,901).

By using the EDC in the DMF/DMSO solvent mixture,, and then it is added drop-wise in the aqueous solution of G5-Ac-AF in the active ester that the described peptide of preparation in the presence of the HOBt is arranged.Response time was respectively 2 hours and 3 days.Amidatioon occurs in mainly that (for example obtain the product of monoamidesization with the A model reaction of the allyl amine of 1.1eq. in DMSO, purity is 67% (HPLC) on the carboxylic acid ester groups of acyl group caproic acid coupling part.ESI-MS?m/z?1282[M+H] ⁺)。The PAMAM dendritic macromole of the partial acetylation of puting together by membrane filtration and dialysis purification and AlexaFluor and RGD peptide G5-Ac-AF-RGD.Conjugate ¹H NMR is presented at overlapped signal in the aromatic compound district of phenyl ring of AlexaFluor and peptide away from the aliphatic compound signal that dendritic macromole is estimated.Based on the MALDI-TOF mass spectrum quantity of peptide class being calculated as has 2-3 peptide in each dendritic macromole.

MALDI-TOF MS become the functionalisation of surfaces that is widely used in characterizing inhomogeneous functionalized dendritic macromole technology (for example, referring to J.Am.Chem.Soc. such as Woller, 2003,125,8820-8826).

Use Waters TOfspec-2E to write down mass spectrum, use high quality P AD detector to postpone extracting mode operation and to use the BSA in sinapic acid to calibrate.In order to measure the functionalized of the dendritic macromole that contains peptide, from product (m/z 32770[M+H] ⁺) middle deduction raw material (m/z 29650[M+H] ⁺).

The scheme of describing above-mentioned synthetic G5-Ac-AF-RGD is as shown in accompanying drawing 31.

Embodiment 15

The external targeting effect of PAMAM-dendritic macromole-RGD4C peptide conjugate

Measure and express high cell surface α _Vβ ₃The cellular uptake of dendritic macromole in the human umbilical vein cell (HUVEC) of receptor-RGD4C conjugate.In brief, in having replenished the RPMI culture medium of endothelial cell growth factor (ECGF), cultivate the HUVEC cell.Handle cell and monitor picked-up with the G5-Ac-AF-RGD conjugate of variable concentrations by flow cytometry.Shown in accompanying drawing 32, flow cytometric analysis has shown with the dosage-dependency and the saturable of HUVEC cell and has combined.

Also use flow cytometry to test of the combination (referring to accompanying drawing 33) of this conjugate with several different cell lines with different integrin receptor expressions.This conjugate shows the different binding affinities with different cell lines, and wherein the HUVEC cell is thereafter the Jurkat cell most effectively in conjunction with conjugate.Reported human lymphocyte in advance and be Jurkat and had a large amount of integrin receptors and can be in conjunction with RGD 4C peptide (for example, referring to Biochemistry such as Assa-Munt, 2001,40,2373).L1210 mouse lymphocyte system can not be in conjunction with conjugate, and the KB cell only shows the moderate combination.

Obviously, conjugate of the present invention shows variable specificity to the cell line with different cell surface integrin receptor expressions.Confirmed by the observed combination of flow cytometry by the confocal microscope analysis.(0,30,60,100nm) the HUVEC cell of concentration processing and fixing with paraformaldehyde (p-formaldehyde) is redyed with DAPI pair cell nuclear with G5-AF-RGD4C in washing.Apparent in the relevant performance of confocal microscope image from accompanying drawing 34, picked-up increases with the increase of conjugate concentration.Add free peptide inhibition HUVEC cellular uptake conjugate and reach significance level, show receptor-mediated conjugate picked-up (referring to accompanying drawing 35).

In order to determine multivalence interacts whether show stronger combining when interacting relatively with unit price, use BIAcore instrument (BIAcore AB, Uppsala, Sweden) to protein (the Chemicon International of human beta 2 integrin alpha v β 3 purification, Inc.Temecula, CA) binding affinity of monitoring G5-Ac-AF-RGD4C conjugate and RGD4C peptide.By using BIAevaluation 3.2 software BIAcore AB) the bivalence combination model is carried out overall match analyze the data that two kinds of analytes are obtained.According to dissociating and the ratio (k of association rate constant _Off/ k _On) calculated equilibrium dissociation constant (K _D).Free RGD4C peptide and human beta 2 integrin alpha v β ₃Combination reach the maximum combined value of 10RU very apace.On the contrary, reach the maximum combined value of about 1500RU more slowly in conjunction with G5-Ac-AF-RGD4C conjugate speed.Two kinds of analytes show different dissociation rate (off-rates).Free RGD4C peptide dissociates from part in service test buffer washing process fast.About slowly 522 times of the specific ionization peptide that dissociates of nanodevice.Therefore, the invention provides multi-functional dendritic macromole, wherein the multiple peptide on single dendritic macromole puts together the result in conjunction with the efficacy exertion synergism.

Therefore, the invention provides PAMAM-dendritic macromole RGD4C peptide conjugate.In certain embodiments, dendritic macromole is by express alpha _Vβ ₃The cellular uptake of receptor.Therefore, in certain preferred aspects, the dendritic macromole conjugate is used to make preparation and/or chemotherapeutics to be oriented to the tumor vascular system that blood vessel takes place.

All open source literatures and the patent mentioned in the above-mentioned description are incorporated herein by reference.Various modification and variation to method and system of the present invention under the situation that does not break away from essence of the present invention and scope are apparent to those skilled in the art.Although described the present invention, it should be understood that the present invention who asks for protection should excessively not be limited to this class specific embodiment in conjunction with concrete preferred embodiment.In fact, the conspicuous modification that is used to implement mode of the present invention of various equivalent modifications is belonged to scope of the present invention.

Claims (89)

1. compositions; comprise dendritic macromole; described dendritic macromole comprises the 5th generation (G5) polyamidoamine (PAMAM) of partial acetylation, poly-propylamine (POPAM) or PAMAM-POPAM dendritic macromole, and described dendritic macromole comprises two or more response locations that are used to put together functional group.

2. the described compositions of claim 1, wherein said dendritic macromole comprises two or more functional groups, and wherein said functional group is selected from the group that therapeutic agent, targeting agent, preparation and biological monitoring agent are formed.

3. the described compositions of claim 2, at least a and described dendritic macromole in the wherein said functional group is puted together by ester bond.

4. the described compositions of claim 2, wherein said therapeutic agent comprises methotrexate.

5. the described compositions of claim 2, wherein said targeting agent comprises folic acid.

6. the described compositions of claim 2, wherein said targeting agent comprises the RGD peptide.

7. the described compositions of claim 2, wherein said preparation comprises fluorescent agent.

8. the described compositions of claim 7, wherein said fluorescent agent comprises Fluorescein isothiocyanate.

9. the described compositions of claim 7, wherein said fluorescent agent comprises 6-TAMARA.

10. the described compositions of claim 4, wherein said methotrexate and described dendritic macromole are puted together by ester bond.

11. the described compositions of claim 1, wherein said dendritic macromole comprise 2-20 response location.

12. the described compositions of claim 2, wherein said dendritic macromole and described functional group put together.

13. the described compositions of claim 12, wherein said puting together comprises covalent bond, ionic bond, metallic bond, hydrogen bond, van der Waals' bond, ester bond or amido link.

14. the described compositions of claim 2, wherein said therapeutic agent comprise chemotherapeutics, resist-cause the tumor agent, anti-angiogenicization agent, tumor inhibitor, antimicrobial or comprise the proteic expression of nucleic acids construct of coding treatment.

15. the described compositions of claim 14, the protected base protection of wherein said therapeutic agent.

16. the described compositions of claim 15, wherein said protecting group are selected from protecting group to photo-labile, to unsettled protecting group of radiation and group that the unsettled protecting group of enzyme is formed.

17. the described compositions of claim 1, wherein said dendritic macromole comprise protected core diamidogen.

18. the described compositions of claim 1, wherein said response location comprises primary amine groups.

19. compositions; comprise dendritic macromole; described dendritic macromole comprises G5PAMAM, POPAM or the PAMAM-POPAM dendritic macromole of partial acetylation; described dendritic macromole further comprises one or more functional groups, and described one or more functional groups are selected from the group that therapeutic agent, targeting agent and preparation are formed.

That 20. the described compositions of claim 19, wherein said therapeutic agent comprise is anti--cause the tumor agent.

21. the described compositions of claim 19, wherein said therapeutic agent comprises chemotherapeutics.

22. the described compositions of claim 19, wherein said therapeutic agent comprises methotrexate.

23. the described compositions of claim 19, wherein said therapeutic agent comprises tritium.

24. the described compositions of claim 19, wherein said targeting agent comprises folic acid.

25. the described compositions of claim 19, wherein said targeting agent comprises the RGD peptide.

26. the described compositions of claim 19, wherein said preparation comprises fluorescent agent.

27. the described compositions of claim 26, wherein said fluorescent agent comprises Fluorescein isothiocyanate.

28. the described compositions of claim 26, wherein said fluorescent agent comprises 6-TAMARA.

29. the described compositions of claim 22, wherein said methotrexate and described dendritic macromole are puted together by ester bond.

30. the described compositions of claim 19, wherein said therapeutic agent are selected from chemotherapeutics, resist-cause the tumor agent, anti-angiogenicization agent, tumor inhibitor, antimicrobial and comprise the group that the proteic expression of nucleic acids construct of coding treatment is formed.

31. the described compositions of claim 19, the protected base protection of wherein said therapeutic agent.

32. the described compositions of claim 31, wherein said protecting group are selected from protecting group to photo-labile, to unsettled protecting group of radiation and group that the unsettled protecting group of enzyme is formed.

33. the described compositions of claim 21, wherein said chemotherapeutics is selected from platinum complexes, verapamil, podophyllotoxin, carboplatin, procarbazine, chlormethine, cyclophosphamide, camptothecine, ifosfamide, melphalan, chlorambucil, busulfan (bisulfan), nitroso ureas (nitrosurea), amycin, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin (plicomycin), mitomycin, bleomycin, etoposide, tamoxifen, paclitaxel, taxol, transplatinum, 5-fluorouracil, vincristin, the group that vinblastine and methotrexate are formed.

34. the described compositions of claim 20 wherein saidly resist-causes the tumor agent and comprises antisensenucleic acids.

35. the described compositions of claim 34, wherein said antisensenucleic acids comprise the complementary sequence with the RNA of oncogene.

36. the described compositions of claim 35, wherein said oncogene are selected from the group that abl, Bcl-2, Bcl-xL, erb, fms, gsp, hst, jun, myc, neu, raf, ras, ret, src and trk form.

37. the described compositions of claim 30, wherein said nucleic acid coding are selected from the protein of the group of tumor suppressor protein, cytokine, receptor, apoptosis inducers and differentiation agent composition.

38. the described compositions of claim 37, wherein said tumor suppressor protein are selected from the group that BRCA1, BRCA2, C-CAM, p16, p21, p53, p73, Rb and p27 form.

39. the described compositions of claim 37, wherein said cytokine are selected from the group that GMCSF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN-β, IFN-γ and TNF form.

40. the described compositions of claim 37, wherein said receptor are selected from the group that CFTR, EGFR, estrogen receptor, IL-2 receptor and VEGFR form.

41. the described compositions of claim 37, wherein said apoptosis inducers are selected from the group that AdElB, Bad, Bak, Bax, Bid, Bik, Bim, Harakid and ICE-CED3 protease are formed.

42. the described compositions of claim 19, wherein said therapeutic agent comprises short-decayed radiosiotope.

43. comprising, the described compositions of claim 19, wherein said preparation be selected from ¹⁴C, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ¹²⁵I, ¹³¹I, ¹¹¹Ln, ¹⁵²Eu, ⁵⁹Fe, ⁶⁷Ga, ³²P, ¹⁸⁶Re, ³⁵S, ⁷⁵Se, Tc-99m and ¹⁷⁵The radioactive label of the group that Yb forms.

44. the described compositions of claim 19, wherein said targeting agent are selected from the group that antibody, receptors ligand, hormone, vitamin and antigen are formed.

45. the described compositions of claim 44, wherein said antibody has specificity to disease specific antigen.

46. the described compositions of claim 45, wherein said disease specific antigen comprises tumor specific antigen.

47. the described compositions of claim 44, wherein said receptors ligand are selected from the group that CFTR part, FGFR part, estrogen receptor ligands, FGR2 part, folacin receptor part, IL-2 receptors ligand, glycoprotein, EGFR part and VEGFR part are formed.

48. the described compositions of claim 44, wherein said receptors ligand are folic acid.

49. the described compositions of claim 44, wherein said receptors ligand are the RGD peptide.

50. the method for treatment disease comprises and suffers from or the experimenter of the described disease of susceptible treats the described compositions of claim 19 of effective dose.

51. the described method of claim 50, wherein said disease are neoplastic disease.

52. the described method of claim 51, wherein said neoplastic disease is selected from leukemia, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin, the non-hodgkin's disease, multiple myeloma, idiopathic macroglobulinemia disease, heavy chain disease, solid tumor, sarcoma and cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterus carcinoma, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, durosarcoma, the group that melanoma and neuroblastoma retinal neuroblastoma are formed.

53. change the method for experimenter's tumor growth, comprise:

A) provide the compositions that comprises dendritic macromole, described dendritic macromole comprises G5 PAMAM, POPAM or the PAMAM-POPAM dendritic macromole of partial acetylation, described dendritic macromole further comprises one or more functional groups, and described one or more functional groups are selected from the group that therapeutic agent, targeting agent and preparation are formed; With

B) under the condition that described tumor growth is changed, give described experimenter described compositions.

54. the described method of claim 53, wherein said change comprise the tumor growth that suppresses among the described experimenter.

55. the described method of claim 53, wherein said change comprise the described tumor size that reduces among the described experimenter.

56. the described method of claim 53 wherein comprises the compositions and the chemotherapeutics of dendritic macromole or resist-causes tumor agent co-administered described.

57. the described method of claim 53, wherein said change tumor growth make described tumor to chemotherapy or anti--carcinogenic therapy sensitivity.

That 58. the described method of claim 53, wherein said therapeutic agent comprise is anti--cause the tumor agent.

59. the described method of claim 53, wherein said therapeutic agent comprises chemotherapeutics.

60. the described method of claim 53, wherein said therapeutic agent comprises methotrexate.

61. the described method of claim 53, wherein said therapeutic agent comprises tritium.

62. the described method of claim 53, wherein said targeting agent comprises folic acid.

63. the described method of claim 53, wherein said targeting agent comprises the RGD peptide.

64. the described method of claim 53, wherein said preparation comprises fluorescent agent.

65. the described method of claim 53, wherein said fluorescent agent comprises Fluorescein isothiocyanate.

66. the described method of claim 53, wherein said fluorescent agent comprises 6-TAMARA.

67. the described method of claim 53, wherein said methotrexate and described dendritic macromole are puted together by ester bond.

68. the described method of claim 53, wherein said therapeutic agent are selected from chemotherapeutics, anti--carcinogen, anti-angiogenicization agent, tumor inhibitor, antimicrobial and comprise the group that the proteic expression of nucleic acids construct of coding treatment is formed.

69. the described method of claim 53, the protected base protection of wherein said therapeutic agent.

70. the described method of claim 69, wherein said protecting group are selected from protecting group to photo-labile, to unsettled protecting group of radiation and group that the unsettled protecting group of enzyme is formed.

71. the described method of claim 59, wherein said chemotherapeutics is selected from platinum complexes, verapamil, podophyllotoxin, carboplatin, procarbazine, nitrogen is situated between, cyclophosphamide, camptothecine, ifosfamide, melphalan, chlorambucil, busulfan (bisulfan), nitroso ureas (nitrosurea), amycin, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin (plicomycin), mitomycin, bleomycin, etoposide, tamoxifen, paclitaxel, taxol, transplatinum, 5-fluorouracil, vincristine, the group that vinblastine and methotrexate are formed.

72. the described method of claim 58 wherein saidly resist-causes the tumor agent and comprises antisensenucleic acids.

73. the described method of claim 72, wherein said antisensenucleic acids comprise the complementary sequence with the RNA of oncogene.

74. the described method of claim 73, wherein said oncogene are selected from the group that abl, Bcl-2, Bcl-xL, erb, fms, gsp, hst, jun, myc, neu, raf, ras, ret, src and trk form.

75. the described method of claim 68, wherein said nucleic acid coding are selected from the protein of the group of tumor suppressor protein, cytokine, receptor, apoptosis inducers and differentiation agent composition.

76. the described method of claim 75, wherein said tumor suppressor protein are selected from the group that BRCA1, BRCA2, C-CAM, p16, p21, p53, p73, Rb and p27 form.

77. the described method of claim 75, wherein said cytokine are selected from the group that GMCSF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN-β, IFN-γ and TNF form.

78. the described method of claim 75, wherein said receptor are selected from the group that CFTR, EGFR, estrogen receptor, IL-2 receptor and VEGFR form.

79. the described method of claim 75, wherein said apoptosis inducers are selected from the group that AdElB, Bad, Bak, Bax, Bid, Bik, Bim, Harakid and ICE-CED3 protease are formed.

80. the described method of claim 53, wherein said therapeutic agent comprises short-decayed radiosiotope.

81. comprising, the described method of claim 53, wherein said preparation be selected from ¹⁴C, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ¹²⁵I, ¹³¹I, ¹¹¹Ln, ¹⁵²Eu, ⁵⁹Fe, ⁶⁷Ga, ³²P, ¹⁸⁶Re, ³⁵S, ⁷⁵Se, Tc-99m and ¹⁷⁵The radioactive label of the group that Yb forms.

82. the described method of claim 53, wherein said targeting agent are selected from the group that antibody, receptors ligand, hormone, vitamin and antigen are formed.

83. the described method of claim 82, wherein said antibody has specificity to disease specific antigen.

84. the described method of claim 83, wherein said disease specific antigen comprises tumor specific antigen.

85. the described method of claim 82, wherein said receptors ligand are selected from the group that CFTR part, FGFR part, estrogen receptor ligands, FGR2 part, folacin receptor part, IL-2 receptors ligand, glycoprotein, EGFR part and VEGFR part are formed.

86. the described method of claim 82, wherein said receptors ligand are folic acid.

87. the described method of claim 82, wherein said receptors ligand are the RGD peptide.

88. the described method of claim 53, wherein said tumor is relevant with neoplastic disease.

89. the described method of claim 88, wherein said neoplastic disease is selected from leukemia, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin, the non-hodgkin's disease, multiple myeloma, idiopathic macroglobulinemia disease, heavy chain disease, solid tumor, sarcoma and cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterus carcinoma, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, durosarcoma, the group that melanoma and neuroblastoma retinal neuroblastoma are formed.

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