CN101351225A - Tumor-targeted nanodelivery systems to improve early mri detection of cancer - Google Patents

Abstract

The present invention is in the fields of drug delivery, cancer treatment and diagnosis and pharmaceuticals. This invention provides a method of making antibody- or antibody fragment-targeted immunoliposomes for the systemic delivery of molecules to treat and image diseases, including cancerous tumors. The invention also provides immunoliposomes and compositions, as well as methods of imaging various tissues. The liposome complexes are useful for encapsulation of imaging agents, for example, for use in magnetic resonance imaging. The specificity of the delivery system is derived from the targeting antibodies or antibody fragments.

Description

Improve the tumor-targeted nanodelivery systems that the cancer early mri detects

Background of invention

Invention field

The invention belongs to medicine and transmit treatment of cancer and diagnosis and drug world.The invention provides the method for preparing antibody or antibody fragment targeting immunoliposome, this immunoliposome is used for the molecule that whole body transmits the treatment disease and makes the disease imaging, and disease comprises cancerous tumour.The present invention also provides immunoliposome and compositions, and the method that makes various imaging of tissue.Liposome complex is used for the parcel of preparation, for example, is used for nuclear magnetic resonance.The specificity of transfer system is derived from targeting antibodies or antibody fragment.

Background of invention

For the target of pain that finishes to be caused by disease and suffering, the ability that detects constitutional and metastatic cancer in early days will be main step.The research and development that are used for the cancer target transfer system of gene therapy have represented the possibility that more effectively transmits preparation than present available method.Nuclear magnetic resonance (MRI) can be obtained the 3-dimension anatomic image of organ.These are caused the accurate location of tumor and the vertical and quantitative monitoring of tumor growth and angiogenesis (Gillies, R.J. etc., Neoplasia2:139-451 (2000) in conjunction with the paramagnetism image; Degani, H. etc., Thrombosis﹠amp; Haemostasis89:25-33)).

One of the most frequently used paramagnetism preparation is in the cancer diagnosis

(gadolinium spray Portugal amine) (Mag) (Berlex Imaging, Montville, NJ).Gadolinium is rare earthy element.Because its ion (Gd ⁺⁺) having seven unpaired electronics, it demonstrates paramagnetic feature.Observed radiography enhancing mainly is because Gd in the MRI scanning ⁺⁺Strong influence to hydrogen proton spin dot matrix relaxation time causes.Therefore although free gadolinium is highly toxic, and be unsuitable for clinical use, produced well tolerable property with the chelating of diethylene-triamine pentaacetic acid (DTPA), stable, strong paramagnetic complex.This metallo-chelate is inert in metabolism.Yet behind the intravenous injection gadolinium spray Portugal amine, the meglumine ion separates with hydrophobicity gadopentetic acid salt (gadopentetate), and it is distributed in the outer water of born of the same parents.It can not pass complete blood brain barrier, and therefore not at normal cerebral tissue, accumulation in the encapsulation, postoperative scar etc., and in urine, discharge fast.It has about 1.6 hours mean half-life.About 80% dosage was discharged in urine in 6 hours.

Yet, use existing contrast agent to have significant limitations, comprise that they mainly are based on perfusion and diffusion labelling and glucose and take in.Use these free (non-compound) reagent, in tumor, see variation in the inflammation disease, even (for example have hormonal effects (in mammary gland), modally proved perfusion and diffused in the intercellular space that based on gadolinium with based on the contrast agent of iodine PDG-PET has proved the glucose absorption).Therefore, these contrast agent do not have target tumor specifically.In addition, active optimum process can not make a distinction with virulent usually, for example, the optimum enhancing zone on the breast MRI, chronic pancreatitis is to cancer of pancreas.Also exist little tumor can not fully take in these reagent, and therefore poor sensitivity and lack earlier detection, this is key especially in the disease as pulmonary carcinoma.It also can not detect solitary pulmonary nodule or pleural nodulations.Therefore, need be used for such reagent is sent to intravital particular organization, for example, be sent to the mechanism of tumor tissues and metastatic tumor.

The invention summary

In one embodiment, the invention provides the method for preparing antibody or antibody fragment target cationic immunoliposome complex, it comprises preparation antibody or antibody fragment, antibody or antibody fragment mixed with cationic-liposome form the cation immunoliposome, wherein antibody or antibody fragment are not chemically being puted together cationic-liposome, and the cation immunoliposome mixed with preparation form antibody or antibody fragment target cationic immunoliposome complex.The example antibody fragment that is used for the present invention's practice comprises strand Fv fragment, as anti-TfR strand Fv (TfRscFv) and anti-HER-2 antibody or antibody fragment.In other embodiment, this method further comprise with the cation immunoliposome with comprise K[K (H) KKK] peptide of 5-K (H) KKC (HoKC) (SEQ ID NO:1) peptide mixes.

Suitably, (w: ratio w) was mixed antibody or antibody fragment with described cationic-liposome with about 1: 20 to about 1: 40.Suitably, cationic-liposome comprises the mixture of two oleoyl front three ammonium phosphate and DOPE and/or cholesterol; Or the mixture of dimethyl two (octadecyl) ammonium bromide and DOPE and/or cholesterol.

In other embodiment, with about 0.1: 10 to about 0.1: 35 ratio (mg preparation: μ g liposome), about suitably 1: 14 to about 1: 28 (the mg preparation: μ g liposome), or about 1: 21 (mg preparation: μ g liposome) the cation immunoliposome is mixed with preparation.The example preparation that is used for the present invention's practice includes, but not limited to nuclear magnetic resonance (MRI) agent, as gadolinium, and gadolinium spray Portugal amine, iopamidol and ferrum oxide.In addition, can also use the barium that is used for CT, iodine and saline preparation are used for PET's ¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG) and other preparations.

The present invention also provides the cation immunoliposome complex that makes by method of the present invention and has comprised cationic-liposome, the antibody of antibody or antibody fragment and preparation or antibody fragment target cationic immunoliposome complex, wherein antibody or antibody fragment are not chemically being puted together with described cationic-liposome.

In further embodiment, the invention provides and make organ or tissue's imaging method, and be used to distinguish patient's benign tissue/disease and cancerous tissue/disease, be included in and carry out before the imaging cation immunoliposome complex of the present invention being delivered medicine to the patient.Can carry out administration by any approach, for example, intravenous administration, intramuscular administration, intradermal administration, eye drops, intraperitoneal administration, administration in the tumor, administration or subcutaneous administration in the intranasal administration, brain.Suitably, using the tissue of method of the present invention and complex imaging is cancerous tissue, comprises carcinous metastatic tumor.

The present invention also provides the method for the patient's that the tumor tissues imaging that makes the patient who suffers from cancer and treatment suffer from cancer tumor tissues, comprises that cation immunoliposome complex of the present invention is delivered medicine to the patient to be made the tumor tissues imaging and anticarcinogen is delivered medicine to the patient and treat tumor tissues.The example anticarcinogen comprises nucleic acid, gene, and protein, peptide, micromolecule, chemotherapeutics, as Docetaxel, mitoxantrone and gemcitabine, and antisense oligonucleotide or siRNA.

Other embodiments of the present invention are that those of ordinary skills know.

The accompanying drawing summary

Figure 1A and 1B have shown the tumour-specific targeting of the CaPan-1 original position metastasis model of TfRscFv-liposome-DNA nano-complex.Identical tumor nodule presents intensive beta galactosidase expression among the 1B in the liver among the 1A shown in the arrow.The 1A=postmortem; Tissue after the dyeing of 1A=beta galactosidase.

Fig. 2 A-2C has shown with after the transfection of TfRscFv-Lip-Mag nano-complex, K564 cells in vitro MRI imaging.The transfection of 1A=time dependence.The value that provides is a relative intensity.1B=demonstrates relative intensity with comprising in the complex

The variation of content (in μ l).The 1C=TfRscFv-Lip-Mag nano-complex is to free

The comparison of relative intensity.Roundlet in all images is the localized labelling of sample.

Fig. 3 A-I has shown the MR imaging of using part-liposome-Mag nano-complex to improve in two various cancers models.3A, D and G have shown the MRI signal difference between big pancreas tumor in situ (arrow) (behind the surgery implantation tumour 4 months) free contrast agent of medium-sized vein drug administration by injection and the TfRscFv-Lip-Mag complex.3B, E and H have shown the similar effect in second mice that has subcutaneous pancreas tumor and littler abdominal part pancreas tumor (arrow).3C, F and I are the images that has the 3rd animal of subcutaneous tumor of prostate (arrow), and wherein identical effect is tangible.

Fig. 4 A-C has not shown and has contained

The SPM phase image of liposome.Each comfortable 1.68V, the set-point of 1.45V and 1.35V has obtained 4A, the image that shows among 4B and the 4C.Corresponding the differing of not being obedient to substrate and mechanically being obedient between the liposome is-3.5 ° ,+8 ° and+40 °.Along with the reduction of setting value, SPM interaction most advanced and sophisticated and liposome changes to repulsion from attraction.

Fig. 5 A-C has shown liposome

(Lip+Mag) SPM and SEM image.5A is a liposome

Particulate atomic force microscope topography.SPM phase image (setting value=1.6) (5B) has similar contrast with 15keV SEM (TE) [transmission mode electronic detector] image (5C), although produce by diverse complementary physical principle.

Fig. 6 A and 6B have shown the SPM landform and the phase imaging of TfRscFv+Lip+Mag nano-complex.6A is 15keV SEM (TE) [transmission mode electronic detector] image of whole nano-complex.The low power image of 6B=field.The zone that frame gets up is the image among the 6A.

The section ratio that Fig. 7 A and 7B have shown SPM landform and magnetic phase image in the lift mode that uses the 25-nm height displacement.7A is the SPM landform/magnetic phase image of whole TfRscFv-Lip-Mag nano-complex.The appearance of the quadripole increment signal that is made of magnetic interaction in attraction and the repulsion face among the 7B has shown that this interactional inducement is the inhomogeneous annular spread of the Magnevist in the NDS, and is consistent with SEM and non magnetic SPM phase image.

Fig. 8 A-8H has shown the MR imaging of using part-HK-liposome-Mag nano-complex to improve in two kinds of various cancers models.People's breast carcinoma MDA-MB-435 (Fig. 8 E-8H) and PC-3 (DU145) (Fig. 8 A-8D).

Fig. 9 A-C has shown the CaPan-1 Subcutaneous tumor of TfRscFV-HK-liposome-Mag nano-complex and the tumour-specific targeting of original position metastasis model.

Figure 10 has shown Dynamic MRI, and this MRI has shown the intensity of using the Mag raising that is transmitted by complex of the present invention in the cancer of pancreas model, compares with free Mag.

Figure 11 A-11C has shown the MR imaging by the cancer of pancreas transfer of the Mag of containing complex of the present invention.

Figure 12 A-12E has shown that lung shifts in the MR imaging the bigger enhancing by the Mag of containing complex of the present invention.

Figure 13 A-13D has shown in the MR imaging that the renal cell carcinoma lung shifts the bigger enhancing by the Mag of containing complex of the present invention.

Figure 14 A-14D has shown the higher detection sensitivity of MR imaging that shifts by renette cell carcinoma lung, utilizes the Mag of containing complex of the present invention.

Figure 15 A-15B has shown the MR imaging by the very little metastatic tumor of the Mag of containing complex of the present invention, has proved the sensitivity of complex of the present invention.

Figure 16 has shown the section of shifting tissue, has confirmed detection/imaging of using the Mag of containing complex of the present invention to see by MRI.

Figure 17 has shown the amplification of the higher multiple of Figure 16.

Figure 18 A-18F has shown the visceral pleura MR imaging of transfer down by the Mag of containing complex of the present invention.

Figure 19 A-19B has shown the B by the Mag of containing complex of the present invention ₁₆/ F ₁₀The detection that the melanoma lung shifts.

Detailed Description Of The Invention

The present invention has satisfied an important demand by the nano-complex that is provided for the preparation whole body is sent to target tissue such as tumour, namely, be used for tumour to sensitivity and the tumor cell specific of the raising of the earlier detection of benign tissue and antidiastole, preparation such as magnetic resonance imaging (MRI) agent, such as gadolinium, Gadopentetate Dimeglumine (

), and Iopamidol, iron oxide; The barium that is used for CT, iodine and salt solution preparation; Be used for PET's¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG) and other preparations. Scanning electron microscopy (SEM) and Scanning Probe Microscopy (SPM) (Wolfert, M.A. etc., Human Gene Therapy 7:2123-2133 (1996); Dunlap, D.D. etc., Nucleic Acids Research 25:3095-3101 (1997); Kawaura, C. etc., FEBS Letters 421:69-72 (1998); Choi, Y.H. etc., Human Gene Therapy 10:2657-2665 (1999); Diebel, C. E. etc., Nature 406:299-302 (2000); Rasa, M. etc., J.Coll.Interface Sci 250:303-315 (2002)) for observing these physical arrangement and sizes with the nano-complex of preparation. In the situation of gadolinium, gadolinium is a kind of high atomicity element with large magnetic moment, can develop these features with various approach and improve contrast among SEM and the SPM. Proved that in this discovery that presents liposome nano-complex of the present invention has wrapped up preparation veritably, as

And the intravenous administration of these compounds forms the tumor imaging that improves. The invention provides the testing result of unexpected and surprising very little metastatic tumor, comprise that the pleura in the lung shifts, and differentiate optimum and ability cancerous tissue.

In one embodiment, the invention provides the cancer target transfer system, comprise contrast preparation, for example magnetic resonance imaging (MRI) contrast preparation. U.S. publication application No.2003/0044407 (its disclosure all being incorporated herein by reference at this) discloses those and has wrapped up the cationic-liposome of the nano-scale of different reagent. What modify these surface of liposome is targeted molecular, and it can be part, such as folate or transferrins, or for antibody or the antibody fragment of cell surface receptor. The having of part/antibody on the liposome helps compound and enters in the cell, by the receptors bind targeted molecular, then the compound of combination is by receptor-mediated encytosis internalization, encytosis is a kind of efficient internalization approach (Cristiano, R. J. and Curiel, D.T., Cancer Gene Therapy 3:49-57 (1996); Cheng, P.W., Human Gene Therapy 7:275-282 (1996)). This modification of liposome causes them not only can be optionally their Payload to be sent to tumour cell, and has improved the transfection efficiency of liposome. TfR (TfR) level obtains raising at various cancerous tissues, comprises the oral cavity, prostate, mammary gland and pancreas (Keer, H.N. etc., Journal of Urology 143:381-385 (1990); Rossi, M.C. and Zetter, B.R., Proc.Natl.Acad.Sci. (USA) 89:6197-6201 (1992); Elliott, R.L. etc., Ann.N.Y.Acad.Sci.698:159-166 (1993); Thorstensen, K. and Romslo, I., Scand.J.Clin.Lab.Investig. (augmenting) 215:113-120 (1993); Miyamoto, T. etc., Intl.J.Oral Maxillofacial Surg. 23:430-433 (1994); Ponka, P. and Lok, C.N., Int ' 1.J.Biochem. Cell Biol.37:1111-1137 (1999)). In addition, recirculation (Ponka in the internalization process of TfR in fast-developing cell such as cancer cell, P. and Lok, C.N., Int ' 1.J.Biochem.Cell Biol.31:1111-1137 (1999)), therefore help the absorption of the nano-complex of these transferrins targets, even in the cancer cell that the TfR level does not have to raise therein. In suitable embodiment, nano-complex described herein uses anti-TfR single chain antibody fragments (TfRscFv) as targeting moiety (Hanynes, B.F. etc., J.Immunol.127:347-351 (1981); Batra, J.K. etc., Molecular ﹠Cellular Biology 11:2200-2205 (1991)). TfRscFv contains complete antibody combining site, is used for the epitope (Batra, J.K. etc., Molecular﹠Cellular Biology 11:2200-2205 (1991)) of monoclonal antibody 5E9 identification TfR. The liposome target is had in the cancer cell of rising TfR level, TfRscFv has the advantage that is better than Tf molecule self or complete Mab: 1) size of scFv (28kDa) is than little many of Tf molecule (80kDa) or parent Mab (155kDa). Therefore scFv-liposome-DNA compound presents and infiltrates better in the distinctive little capillary of entity tumor. 2) less scFV has the advantage of practice for the large-scale production that clinical testing needs. 3) scFV is recombinant molecule and is not the blood product the same with Tf, and therefore do not have by blood with the danger of the potentially contaminated that causes of pathogen. 4) do not have the Fc fragment of Mab, eliminated the result (Jain, R.K. and Baxter, L.T., Cancer Res.48:7022-7032 (1988)) by the non-antigentic specificity combination of Fc acceptor. Like this anti--TfR single-chain antibody molecule can be preferentially with the cationic-liposome of intravenous administration-DNA nano-complex target tumor (referring to, U.S. publication application No.2003/0044407; Xu, L. etc., Molecular Medicine 7:723-734 (2001); Xu L etc., Molecular Cancer Therapeutics 1:337-346 (2002)). Will

(Mag) be wrapped in the sensitivity that has given raising in such cancer target nano-complex and the advantage that detects metastases and cancer diagnosis. Gadolinium, Gadopentetate Dimeglumine (

), Iopamidol, iron oxide; The barium that is used for CT, iodine and salt solution preparation; Be used for PET's¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG) and other preparations, and any other contrast preparation commonly used known to persons of ordinary skill in the art, and any contrast preparation that in the future remains to be researched and developed or preparation are (for example, be used for MRI, CT, PET, SPECT etc.) also can be wrapped in the immunoliposome of the present invention.

By simple and effectively non-chemically conjugation methods make cationic liposome complex according to antibody of the present invention or antibody fragment target, in the method with required compound composition with the order of the ratio that limits and restriction mix (referring to, U.S. publication application No.2003/0044407). Resulting compound is the same effective with the similar compound that wherein antibody or antibody fragment and liposome or polymer chemistry are puted together, or more effective. Term " immune complex ", " immunoliposome ", " compound ", " nano-complex ", " immune nano compound ", " liposome complex " is used interchangeably, and all is used for representing cationic-liposome of the present invention.

Complete antibody or antibody fragment can be used for preparing compound of the present invention. In suitable embodiment, use antibody fragment. Preferably, antibody fragment is the Single-Chain Fv Fragment of Murine of antibody. A kind of preferred antibody is anti-TfR monoclonal antibody, and preferred antibody fragment is based on the scFv of anti-TfR monoclonal antibody. Suitable anti-TfR monoclonal antibody be 5E9 (referring to, for example, Hayes, B.F. etc., " Characterization of a Monoclonal Antibody (5E9) that Defines a Human Cell Surface Antigen of Cell Activation " (sign of the monoclonal antibody (5E 9) of the human cell surface antigen of restriction cell-stimulating), J. Immunol.127:341-352 (1981); Batra, J.K. etc., " Single-chain Immunotoxins Directed at the Human Transferring Receptor Containing Pseudomonas Exotoxin A or Diphtheria Toxin:Anti-TFR (Fv)-PE40 and DT388-Anti-TFR (Fv) " (for the monochain immunotoxin of the people's transfer receptor that contains ETA or diphtheria toxin: anti--TFR (Fv)-PE40 and DT388-be anti--TFR (Fv)) and, Mol.Cell.Biol.11:2200-2205 (1991); Its disclosure is incorporated herein by reference). ScFv based on 5E9 antibody contains complete antibody combining site, is used for the epitope of the TfR of this Mab identification, as the single polypeptide chain of about molecular weight 26,000. Form scFv from composition VH and the VL variable domains of heavy chain and light chain separately by connection, use the suitably connection peptide of design, its bridge joint the C-end of first variable region and the N-of second variable region hold, order is VH-attachment-VL and VL-attachment-VH. Another preferred antibody is anti--HER-2 monoclonal antibody, and the scFv of another preferred antibody fragment is based on anti--HER-2 monoclonal antibody.

In suitable embodiment, cysteine partly is added into the C-end of scFv. Although do not wish to be bound by theory, thinking provides the cysteine of free sulfhydryl groups can improve complex formation between antibody and the liposome, for example, and by electric charge-charge interaction. Use or without cysteine, can be in E.coli inclusion body marking protein, then refolding produces the antibody fragment of activity form.

If do not wish in complex formation immunoliposome stable on the usage space, first step of preparation compound comprises that the composition with cationic-liposome or liposome or little polymer mixes (referring to embodiment and U.S. publication application No.2003/0044407 herein) with antibody or the antibody fragment of selection. Multiple cationic-liposome can be used in the preparation of compound of the present invention. Disclosed PCT application WO99/25320 has described the preparation of several cation liposome. The example of desirable liposome comprises the mixture that contains two oleoyl front three ammonium phosphate (DOTAP) and DOPE (DOPE) and/or cholesterol (chol); Or those of the mixture of DDA (DDAB) and DOPE and/or chol. For specific target cell type, the ratio that can change lipid is come the absorption efficient of optimization treatment molecule. Liposome can comprise one or more cation lipids and one or more mixtures neutral or auxiliary lipid. Cation lipid is about 1 with the desired proportions of neutral or auxiliary lipid: (0.5-3), and preferred 1: (1-2) (mol ratio).

The present invention also provides the target cationic polymer that is used for transmitting preparation. Suitable polymer is can mediated dna to shrink and can mediate DNA that endosome discharges in conjunction with cationic polymer. Preferred polymer is polymine. Other useful polymer comprise polylysine, the tree-shaped polymer of nucleoprotamine and daiamid.

Antibody or antibody fragment are in conjunction with the antibody on target cell surface or antibody fragment, and the receptor of preferred combination differential expression on target cell.In room temperature and cationic-liposome or polymer mixed, and protein: the ratio of lipid is that (w: w), or the ratio of protein polymer was about 0.1: 1 to 10: 1 (mol ratio) in about 1: 20 to about 1: 40 with antibody or antibody fragment.

Make antibody or antibody fragment and liposome or polymer in a bit of time of incubated at room, about usually 10-15 minute, then mixture is mixed with the therapeutic agent or the diagnostic agent of selection.Can comprise gene, high-molecular-weight DNA (genomic DNA), plasmid DNA, antisense oligonucleotide with the example of antibody and compound treatment molecule of liposome or therapeutic agent, peptide, ribozyme, nucleic acid (comprising siRNA and antisense), virion, immunomodulator, protein, micromolecule and chemical reagent.Preferred treatment molecule comprises coding p53, the gene of Rb94 and Apoptin.RB94 is the variant of retinoblastoma suppressor gene.Apoptin is the gene of an inducing apoptosis of tumour cell.In another preferred embodiment, reagent is antisense oligonucleotide or siRNA molecule, as HER-2 antisense or siRNA molecule.The third preferred reagent is diagnostic imaging agent, as the MRI preparation, as the Gd-DTPA agent.Other preparations include, but not limited to gadolinium, gadolinium spray Portugal amine (

), iopamidol, ferrum oxide; The barium that is used for CT, iodine and saline preparation; With the 18F-2-deoxidation that is used for PET-2-fluoro-D-glucose (FDG) and other preparations.If reagent is DNA, as the encode fragment of p53, can be placed on strong constitutive promoter, under the control as RSV or CMV promoter.

With antibody or antibody fragment and liposome composition the nmol TL) or (μ g reagent: the ratio nmol total polymer) was mixed with therapeutic agent or diagnostic agent in 1: 10 to 1: 40 with about 1: 10 to 1: 20 (μ g reagent:, then in a bit of time of incubated at room, about 10 to 15 minutes usually.In the scope of about 50-400nm, it is measured to pass through dynamic light scattering as use Malvern Zetasizer 3000 usually for the size of liposome complex.

In one embodiment of the invention, the liposome that is used to form complex is a stabilized liposomes on the space.Stabilized liposomes is wherein to have combined hydrophilic polymer such as PEG, poly-(2-ethylacrylic acid) or poly-(n-N-isopropylacrylamide) liposome (PNIPAM) on the space.When with therapeutic agent or diagnostic agent compound tense, the liposome of modified is particularly useful like this, does not clear out from blood flow by the reticuloendothelium system so fast because they resemble usually without the so suitable liposome of modified.In order to prepare stabilized liposomes complex on the space of the present invention, mixed antibody or antibody fragment, the order of liposome and therapeutic agent or diagnostic agent is opposite with above-mentioned order.In a first step, at first (μ g reagent: the ratio nmol lipid) was mixed with above-mentioned therapeutic agent or diagnostic agent with about 1: 10 to 1: 20 with cationic-liposome.The PEG polymer solution that can accept on the physiology in the buffer is added among this lipoplex, and resulting solution is enough to make the time of polymer in conjunction with liposome complex in incubated at room.Then with antibody or antibody fragment and stabilized liposomes polymer in mixed at room temperature, and protein: the ratio of lipid is about 1: 5 to about 1: 30 (w: w).

Liposome that makes according to the present invention or polymer complex can be mixed with acceptable preparation on the materia medica that is used for vivo medicine-feeding.The complex bound drug can be learned and go up compatible medium or carrier.Can compositions formulated, for example, be used for intravenous administration in people patient, this patient has benefited from the treatment of complex or the administration of diagnosis molecule.The size of complex is suitable, makes can to distribute by whole body behind intravenous administration.Perhaps, can transmit complex by other administration route, as (IT) in the tumor, in sick the damage (IL), aerosol, percutaneous, endoscope, part, intramuscular (IM), Intradermal (ID), ophthalmic (IO), intraperitoneal (IP), (IC) or subcutaneous administration in the intranasal (IN), brain.It is well known in the art being used for the preparation of the preparation that transmits by such method and using the transmission of such method.

In one embodiment, administration comprises the liposome (or polymer) of antibody or antibody fragment targeting and the compositions of therapeutic agent complex, to realize people's gene therapy.The therapeutic agent component of complex comprises the therapeutic gene under the suitable regulating and controlling sequence control.Transmit the gene therapy that the antibody of the nucleic acid contain coding wt p53 or RB94 or antibody fragment target liposomes or polymer complex realize being used for various forms of human cancers by whole body.Complex is the tumor cell of targeting and sensitization constitutional and metastatic tumo(u)r specifically, with irradiation and/or chemotherapy in vitro and in vivo.

Can be at selection and the ratio of target cell type by lipid, the ratio of antibody or antibody fragment and liposome, the ratio of antibody or antibody fragment and liposome and therapeutic agent or diagnostic agent, and the selection of antibody or antibody fragment and therapeutic agent or diagnostic agent comes the optimization complex.

In one embodiment, target cell is a cancerous cell.Although any tissue with malignant cell growth can be a target, head and neck, mammary gland, prostate, pancreas, brain, comprise glioblastoma, cervix uteri, lung, liver, liposarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma, retinoblastoma, the uterus, apparatus urogenitalis, the human colorectal cancer of harmonization of the stomach is suitable target.

The complex that makes by method of the present invention can also be used for the targeting non-tumor cell, is used for transmitting treatment molecule or any nucleic acid.Although any normal cell can be an object, preferred cell is dendritic cell, the endotheliocyte of blood vessel, pneumonocyte, mammary glandular cell, medullary cell, thymocyte cell and hepatocyte.But can the disadvantageous benign cell of targeting, as the benign prostatic hyperplasia cell, Huo Yue thyroid cell too, the cell that the lipoma cell is relevant with autoimmune disease, relate to arthritis as generation, lupus, myasthenia gravis, squamous metaplasia, degeneration of macula, cardiovascular disease, neuropathy, as Alzheimer, the B cell of the antibody of abnormal development etc.

Can come the administration complex in conjunction with another kind of therapeutic treatment, as radiotherapy or chemotherapeutics.Can before or after the administration of complex, give therapeutic treatment, or the combination of therapeutic treatment, for example in about 12 hours to about 7 days.Chemotherapeutics comprises, for example, amycin, 5-fluorouracil (5FU), cisplatin (CDDP), Docetaxel (

), gemcitabine (

), paclitaxel, vincaleucoblastine, etoposide (VP-16), camptothecine, actinomycin D, mitoxantrone and ametycin.Radiotherapy/processing comprise γ radiation ( ¹³⁷Cs), X-ray, UV irradiation, microwave, electron emission etc.Other therapeutic agent comprises micromolecule, peptide, protein etc.

Can also diagnostic agent or preparation be sent to targeted cells by liposome or polymer complex.Term " diagnostic agent " and " preparation " are used interchangeably, and all are used for being illustrated in obtaining detecting range estimation, the reagent of imaging or observation after the administration in vivo.Be used for detecting, range estimation, the case method of imaging or inspections and examinations agent and preparation is well known in the art, and comprise, for example, optical imagery, as fluorescence imaging (exometer) or bioluminescence imaging, positron emission fault (PET) scanning, single photon emission computed tomography (SPECT) scanning, nuclear magnetic resonance (MRI), the x-ray, radioactive nucleus thuja acid imaging (for example, gammacamera, computed tomography (CT), quantitative automatic radiography etc.) etc.The example diagnostic agent comprises the electron density material, ferrum, nuclear magnetic resonance agent and radiopharmaceutical.The useful radionuclide of imaging comprises copper, gallium, and indium, the radiosiotope of rhenium and technetium comprises isotope ⁶⁴Cu, ⁶⁷Cu, ¹¹¹In, ⁹⁹MTc, ⁶⁷Ga or ⁶⁸Ga.MRI agent such as Gd-DTPA agent, gadolinium or

(gadolinium spray Portugal amine) (Mag) (Berlex Imaging, Montville, NJ).Disclosed preparation in U.S. patent 5,688,488 such as Low can be used for being introduced into as a reference at this among the present invention.Other preparations (for example, include, but not limited to iopamidol RegionalHealth Limited, Aukland, AU), ferrum oxide; The barium that is used for CT, iodine and saline preparation; Be used for PET's ¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG) and other preparations.

The complex of prepared in accordance with the method for the present invention can be provided with the form of test kit, and this test kit is used for transmitting treatment molecule or diagnosis molecule by the complex whole body.Suitable test kit can comprise suitable containers (or in one container), liposome, antibody or antibody fragment and therapeutic agent or diagnostic agent dividually.Can be to deliver medicine to the patient in suitable order blending ingredients and the reasonable time section after preparation under aseptic condition, about 30 minutes to about 24 hours usually.Preferably provide reagent constituents as solution or as dry powder.Preferably the set of dispense that the solution form is provided is formed in the sterile water for injection and suitable buffer, and Morie osmolarity controlling agent etc. together.

The parcel of preparation and transmission

In specific embodiment, the invention provides the cation lipid nanocrystal composition, wherein one or more preparations are wrapped in the liposome interior, be included in double-deck hydrocarbon chain fragment, compound with inboard and/or outside monolayer/as (for example to link to each other, by electrostatic interaction or chemistry/covalent interaction), or any or whole combinations in these probabilities.Suitably, preparation is wrapped in the inside of liposome and/or and links to each other with inboard and/or outside monolayer.

As used in this, term " diagnostic agent " and " preparation " can obtain detecting range estimation, the reagent of imaging or observation after referring to administration in vivo.The example preparation comprises the electron density material, ferrum, nuclear magnetic resonance agent and radiopharmaceutical.The useful radionuclide of imaging comprises copper, gallium, and indium, the radiosiotope of rhenium and technetium comprises isotope ⁶⁴Cu, ⁶⁷Cu, ¹¹¹In, ^99mTc, ⁶⁷Ga or ⁶⁸Ga.MRI agent such as gadolinium, the Gd-DTPA agent or

(gadolinium spray Portugal amine) (Mag) (Berlex Imaging, Montville, NJ).Disclosed preparation in U.S. patent 5,688,488 such as Low also can be used for being introduced into as a reference at this among the present invention.Other preparations (for example, include, but not limited to iopamidol

RegionalHealth Limited, Aukland, AU), ferrum oxide; The barium that is used for CT, iodine and saline preparation; Be used for PET's ¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG) and other preparations.

As described herein, can be in the course of processing by mix simply one or more preparations and liposome with preparation suitably with liposome complex parcel of the present invention, comprise or compound/link to each other.Can easily measure preparation by those skilled in the art: the proper ratio of liposome complex.For example, the ratio of preparation and liposome complex is about 0.1: 10 to about 0.1: 35 (mg preparation: μ g liposome) suitably, more suitably about 1: 14 to about 1: 28 (the mg preparation: μ g liposome), or about 1: 21 (mg preparation: μ g liposome).

Described in full text, the example that is used to transmit/comprise the desirable cationic-liposome of preparation comprises the mixture that contains two oleoyl front three ammonium phosphate (DOTAP) and DOPE (DOPE) and/or cholesterol (chol); Or those of the mixture of dimethyl two (octadecyl) ammonium bromide (DDAB) and DOPE and/or chol.The ratio that can change lipid is come the absorption efficient of optimization preparation.Liposome can comprise one or more cation lipids and one or more mixture neutral or auxiliary lipid.Cation lipid is about 1 with the desired proportions of neutral or auxiliary lipid: (0.5-3), and preferred about 1: (1-2) (mol ratio).The example of useful various lipid ratios includes, but are not limited in the present invention's practice:

1: 1 mol ratio of LipA DOTAP/DOPE

1: 1 mol ratio of LipB DDAB/DOPE

1: 2 mol ratio of LipC DDAB/DOPE

1: 1 mol ratio of LipD DOTAP/Chol

1: 1 mol ratio of LipE DDAB/Chol

2: 1: 1 mol ratios of LipG DOTAP/DOPE/Chol

2: 1: 1 mol ratios of LipH DDAB/DOPE/Chol

(DOTAP=two oleoyl front three ammonium phosphate, DDAB=dimethyl two (octadecyl) ammonium bromide; The DOPE=DOPE; The chol=cholesterol).

In one embodiment, the invention provides the method that preparation contains the antibody or the antibody fragment target cationic immunoliposome complex of preparation, comprise preparation antibody or antibody fragment; Antibody or antibody fragment mixed forming the cation immunoliposome with cationic-liposome, wherein antibody or antibody fragment are chemically being puted together with cationic-liposome; And the cation immunoliposome mixed with one or more preparations form antibody or antibody fragment target cationic immunoliposome complex.

In suitable embodiment, antibody fragment is a strand Fv fragment, for example, and anti-TfR strand Fv (TfRscFv) and anti-HER-2 antibody or antibody fragment.Described the example of the suitable lipid that is used to prepare the cation immunoliposome that contains preparation at this, and comprised, the mixture of two oleoyl front three ammonium phosphate and DOPE and/or cholesterol; Or the mixture of dimethyl two (octadecyl) ammonium bromide and DOPE and/or cholesterol.Suitably, (w: ratio w) was mixed antibody or antibody fragment and is formed the cation immunoliposome with cationic-liposome with about 1: 20 to about 1: 40.Suitably, with about 0.1: 10 to about 0.1: 35 ratio (mg preparation: μ g liposome), more suitably about 1: 14 to about 1: 28 (the mg preparation: μ g liposome), or about 1: 21 (mg preparation: μ g liposome) the cation immunoliposome is mixed with preparation.

The example preparation comprise described herein and known in the art those.Suitably, preparation is the MRI preparation, as gadolinium, and gadolinium spray Portugal amine, iopamidol is (for example,

RegionalHealth Limited, Aukland, AU), or ferrum oxide; The barium that is used for CT, iodine and salt preparation; Be used for PET's ¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG) and other preparations.

In other embodiments, method of the present invention and immunoliposome complex further comprise with cationic-liposome with comprise K[K (H) KKK] peptide of 5-K (H) KKC (HoKC or HK) (SEQ IDNO:1) peptide mixes.The HoKC peptide carries terminal cysteine to allow to put together maleimide base group.Therefore, when using the HoKC peptide, Liposomal formulation also comprises the N-maleimide-phenylbutyric acid salt-DOPE (MPB-DOPE) of 0.1 to 50 molar percentage of TL, more preferably the 1-10 molar percentage of TL, most preferably 5 molar percentages of TL suitably.According to described preparation HoKC liposome (Yu before, W etc., Enhanced transfectionefficiency of a systemically delivered tumor-targetingimmunolipoplex by inclusion of a pH-sensitive histidylatedoligolysine peptide (improving the transfection efficiency of the cancer target immunity lipoplex of whole body transmission) by comprising the few lysine peptide of pH-sensitivity histidyl-salinization, Nucleic AcidsResearch 32, e48 (2004)).

In further embodiment, the invention provides antibody or antibody fragment target cationic immunoliposome complex, it comprises cationic-liposome, antibody or antibody fragment, with one or more preparations, wherein antibody or antibody fragment are not chemically being puted together with cationic-liposome.With antibody or antibody fragment (for example by the interaction between antibody or antibody fragment and the liposome, static, the interaction that Fan Dehuashi power or other are puted together non-chemically) links to each other suitably with liposome, suitably by cysteine residues on antibody or the antibody fragment and the interaction between the surface of liposome.Usually, junctional complex or sept molecule (for example, polymer or other molecules) are not to be used for connecting antibody and liposome.Preparation can be wrapped in the cationic-liposome, be included in the hydrocarbon chain fragment of cationic-liposome, with cationic-liposome, or the inboard of its any combination or outside monolayer link to each other.Suitably, cation immunoliposome of the present invention is unilamellar liposome (that is, single bilayered), although also can use the multilamellar liposome that comprises several concentric bilayers.Single bilayered cation immunoliposome of the present invention comprises that inside contains water capacity, wherein can wrap up reagent (for example, preparation).They can also comprise single bilayered, and it has hydrocarbon chain fragment (that is, the fat chain fragment of lipid), wherein can contain reagent (for example, preparation).In addition, reagent (for example, preparation) can with the arbitrary of the inboard monolayer of liposome membrane (that is the head group part of lipid) and/or outside monolayer or both be compound or continuous, for example, finish by the electric charge-charge interaction between negative charge preparation and the positive charge cationic-liposome.In further embodiment, reagent (for example, preparation) wrap up/link to each other/can be compound among arbitrary or whole in these zones of cation immunoliposome complex of the present invention.

In further embodiment, the invention provides the organ or tissue's imaging method that makes the patient, be included in and carry out before the imaging cation immunoliposome complex that contains preparation of the present invention being delivered medicine to the patient.Can come administration immunoliposome complex by any required approach, include, but are not limited to, intravenous (IV), oral, the part, by sucking, intramuscular (IM) injection, (IT) injection in the tumor, Intradermal (ID) injection, intraperitoneal (IP) injection, intranasal (IN) injection, ophthalmic (IO) injection, intracranial (IC) injection or other approach.As used in this, the term patient comprises animal patient (for example, non-human mammal, as Canis familiaris L., cat, pig, sheep etc.) and people.To be used for the patient tissue imaging method be well known in the art and include, but not limited to PET scanning, SPECT scanning, MRI imaging etc.Can use method of the present invention and complex to make patient's any tissue or organ imaging.By the targeting part on the modified liposome simply, albumen or molecule that can any overexpression of targeting.

Suitably, method of the present invention is used for making the cancerous tissue imaging of suffering from or being easy to suffer from the patient of cancer.Can use the cancerous tissue of method imaging of the present invention to comprise solid tumor, and metastasis.Method of the present invention can also be distinguished cancerous tissue and non-carcinous (optimum) tissue.

In further embodiment, the invention provides to make and suffer from or cancer-prone patient's tumor tissues imaging and the method for the treatment of, comprise that the administration immunoliposome complex that contains preparation of the present invention makes the tumor tissues imaging, and anticarcinogen is delivered medicine to the patient treat tumor tissues.

Anticarcinogen example that can administration includes, but not limited to micromolecule, protein, peptide and chemotherapeutics, as described herein those, gene, antisense oligonucleotide and siRNA.The example chemotherapeutics includes, but not limited to amycin, 5-fluorouracil (5FU), and cisplatin (CDDP), Docetaxel (

), gemcitabine ( ), paclitaxel, vincaleucoblastine, etoposide (VP-16), camptothecine, actinomycin D, mitoxantrone and ametycin, and Antybody therapy, as monoclonal antibody, for example,

(Genentech, SanFrancisco CA).Being used for the antisense oligonucleotide of the present invention's practice and the example of siRNA molecule comprises, but be not limited to, U.S. the U.S. patent application No.11/520 of publication application No.2003/0044407 and JIUYUE in 2006 application on the 14th, those disclosed in 796 is incorporated herein by reference with its integral body in this disclosure with every piece.Other anticarcinogen comprise peptide, protein and micromolecule (referring to, for example, the U.S. temporary patent application No.60/800 of application on May 15th, 2006,60/844,352 of 163 and 2006 on JIUYUE application in 14, is incorporated herein by reference with its integral body in this disclosure with every piece).Can be (for example with anticarcinogen, chemotherapeutics, micromolecule, gene or antisense or siRNA etc.) link to each other with the cation immunoliposome that also comprises preparation, or with it in different immunoliposomes according to the present invention, or separately transmit by another carrier or transfer system the chemotherapeutics IV injection of normal clinical standard (for example, according to).

In suitable embodiment, method of the present invention is included in different time administrations and comprises that preparation (for example, the MRI preparation is as gadolinium spray Portugal amine) immunoliposome complex and anticarcinogen (that is, can give complex and reagent simultaneously or in the different time).Suitably, administration anticarcinogen before or after comprising the immunoliposome complex of preparation, (for example, before or after the administration cation immunoliposome complex at least 1 hour, before or after at least 6 hours, before or after at least 12 hours, before or after at least 24 hours, before or after at least 48 hours etc.).In further embodiment, the method that makes cancer patient's tumor tissues imaging and treat cancerous tissue further comprises to the patient and gives radiotherapy.

Information contained and that this area obtains easily in according to the present invention, those skilled in the art can easily determine anticarcinogen suitable in the human body (for example, chemotherapeutics, gene, micromolecule, protein, peptide, antisense oligonucleotide or siRNA etc.) dosage and administration time.In addition, by to animal mice for example, rat, such content is estimated in the experiment that Canis familiaris L. carries out or the deduction of other researchs.

The example benefit of using nano immune liposome complex of the present invention (scL and scL-HoKC) to wrap up and transmit preparation comprises because the higher concentration that the cancer target character of complex causes in cancerous tissue.Because complex accumulates in cancerous cell, therefore from the imaging of cancer specificity exist blood vessel to flow and diffuse to the intercellular space difference (as use at present clinical the non-compound free preparation of use see).Also exist cancer that the difference of optimum process is strengthened.Long blood vessel and the delay imaging of organizing half-life permission use complex of the present invention.Complex and method can be used for making the destination organization imaging of different depth.

Therefore, method of the present invention and complex not only cause the signal that improves in the tumor, and cause the higher definition of inside tumor structure.More specifically, can detect less tumor, make more early to obtain detecting also therefore improving and reply/survive.These complex can also be used to distinguishing benign and pernicious tuberosity.When this help to promote to determine begin treatment.At present, owing to uncertainly be virulent or be not virulent, postpone usually to determine whether tuberosity increases.Yet because complex of the present invention is preferred and transfection tumor cell specifically, this can also be as the confirmation of malignant tumor, and for example, whether the lesser tubercle of seeing on lung CT is little malignant tumor.These last 2 is particular importance in pulmonary carcinoma and cancer of pancreas.

The cancer types example that the complex that contains preparation of the application of the invention can solve imaging problem comprises, cancer of pancreas, and earlier detection also makes a distinction with chronic pancreatitis; The metastatic disease of earlier detection pulmonary; Isolatism pulmonary tuberal region is divided into optimum or pernicious; The enhanced little focus area of the MR that improves in the mammary gland is divided into optimum or pernicious.

Complex of the present invention can also be used to confirming: use this transfer system, therapeutic agent will more may enter the specific cancerous cell of patient.That is, contain the fact that the complex of preparation can enter cell, provide the transmission of the therapeutic agent that links to each other with complex of the present invention or other reagent also will enter the sign of these particular cancer cells.

Those skilled in the art will easily learn other the suitable modifications and the improvement that can form methods and applications described herein, and not depart from the scope of the present invention or embodiment that it is any.Described the present invention in detail,, comprised that at this these embodiment just are used for illustrative purposes, be not to be used for limiting the present invention by will more being expressly understood with reference to following embodiment.

Specific embodiments

Embodiment 1

Comprise The immunoliposome complex

Material and method

Cell line

Obtaining the human lymphocyte leukaemia from Lombardi Comprehensive Cancer Tissue Culture core institution is K562.These suspension cells are maintained at additional 10% hot deactivation FBS add the 2mM L-glutaminate, penicillin is among the RPMI1640 of each 50 μ g/ml of streptomycin and neomycin.Produce human pancreatic cancer cell CaPan-1 (from ATCC Manassas, VA obtains) from the adenocarcinoma metastatic of pancreas.It is maintained at contains 4mM L-glutaminate and sodium bicarbonate, replenish 20% non-heat-inactivated FBS, 2mM L-glutaminate and penicillin are among Iscov ' the s Modified Dulbecco ' sMedium of each 50 μ g/ml of streptomycin and neomycin.Initial from extensive metastatic prostate cancer patient's brain injury, produce PC-3 DU145 (ATCC, Manassas, VA).It is maintained at the MEM that contains Earle ' s salt (EMEM) that additional 10% heat-inactivated FBS adds L-glutaminate and aforesaid antibody.

Nano-complex forms

Prepare cationic-liposome (DOTAP:DOPE) (referring to U.S. publication application series No.2003/0044407 according to described ethanol injection method before; XuL etc., MolecularCancer Therapeutics 1:337-346 (2002) is incorporated herein by reference every piece disclosure).When transmitting plasmid DNA, form complete complex (referring to U.S. publication application series No.2003/0044407) with described same way as before.In order to wrap up the preparation that is used for external use, TfRscFv is mixed with liposome with specific ratio, and incubated at room 10 minutes.Will

Add in this solution, mix and once more incubated at room 10 minutes.When being stored in 2-8 ℃, complex was stablized 8 days at least, as using MalvernZetasizer 3000H determined by dimensional measurement.The cumulant of measuring along with this time frame (Z is average) on average is 112.3 ± 4.67 (S.E.), and polydispersity (repeatability of the value in the expression multiple scanning process) is 0.445 ± 0.03.The acceptable size scope of nano-complex is about 20 to 1000nm, is about 50 to 700nm suitably, and more suitably about 100 to 500nm.For in-vitro transfection, before transfection, the 2ml serum-free medium is added in the complex.For using in the body, use said method with the 1mg preparation than 0.33-1.17 μ gTfRscFv than the ratio of 10-35 μ g liposome form complex (suitably the 1mg preparation than 0.5 to 1.0 μ g TfRscFv than 14-28 μ g liposome, most suitably the 1mg preparation than 0.71 μ g TfRscFv than 21 μ g liposomees).Be used for using in the body and during preparation, add the final concentration of glucose to 5%.

In-vitro transfection

For transfection suspension cell K562, will use except 15 * 10 in the 4.0ml cumulative volume culture medium of all fill-ins of serum ⁶Cell places 100mm ²In tissue culture's ware.2ml is contained different content

Above-mentioned transfection solution add in the cell suspending liquid.Flat board is hatched at 37 ℃, softly rock, the time span (up to 90 minutes) that provides in lasting result's part, after this with cell 4 ℃ of gentle precipitations (600 * g7 minutes) in the 0.5ml microcentrifugal tube, and remove any excessive transfection solution three times and place with the washing of 10ml serum-free medium and wet on ice until imaging.

The in-vivo tumour targeting

In order to measure the tumor-selective targeting of TfRscFv-Lip nano-complex to constitutional and metastatic tumo(u)r, used the original position metastasis model, this model has used human pancreatic cancer cell CaPan-1.Be suspended in Matrigel by injection ^TMIn the collagen basement membrane matrix (BDBiosciences) 1 * 10 ⁷The subcutaneous xenotransplantation tumor of CaPan-1 in the female athymic mouse of CaPan-1 cell induction.After about eight weeks, collect tumor and prepare the single-cell suspension liquid of tumor.To be suspended in Matrigel equally ^TMIn 1.2-1.5 * 10 ⁷Injection cell is to the pancreas of female athymic mouse surgical exposure.In operation five weeks of back, will carry the complex of LacZ gene at 24hr angular vein injection 3X (40 μ g DNA/ injection).After 60 hours, express painted organ (Xu, L etc., Human Gene Therapy 10:2941-2952 (1999)) with the existence of described method inspection transfer before sacrifice of animal and the use with for beta galactosidase.

The MRI imaging

For external MRI imaging, the cell precipitation in the microcentrifugal tube is placed the center of Magnet.(Varian Inc, Palo Alto CA) carry out the MR imaging to use 4.7T lateral aperture NMR machine in Howard University.The imaging experiment scheme comprises many thin slices T1-weighting spin echo imaging sequence and saturated recovery sequence.For T1-weighted imaging technology, the repetition time (TR) is 1000ms, and ET (TE) is 13ms.Use T1-weighting spin echo imaging technique and confirm that forward becomes image intensifying.To use the saturated-recovery MR order of different ETs to be used for T1 and to measure.The slice thickness of image is 0.5mm.Used RF volume is a 30mm monocycle volume.The RF volume is as the RF emitter and receiver.The RF pulse is optionally 5ms sinc pulse.The quantity of phase coding step is 256.The visual field is 15mm * 15mm.The image area of selecting in the research is positioned at the center of RF volume, is used for the RF uniformity.Obtain the MR image in the cross-wise direction of microcentrifugal tube.The height of cell precipitation is 12mm.The scope of multislice images covers whole precipitate.The image of center section is used for research, and this image is not subjected to owing to influence the influence of the anamorphose that causes from the sensitivity on air-precipitation border.Use Varian image preview software measurement image intensity.Obtain signal from the target area, it enough covers 2/3rds images of taking from each microcentrifugal tube greatly.Contrast be will be used for from the sedimentary relative image intensity of these pipes and evaluation and T1 measurement strengthened.

For studying in the body, use the mice that has CaPan-1 tumor in situ or the subcutaneous xenotransplantation tumor of DU145.Induce the CaPan-1 tumor as mentioned above.By 7 * 10 among the subcutaneous vaccination Matrigel ⁶Cell is induced the DU145 tumor.Carry out these researchs in Georgetown University.Anesthesia is treated the animal of imaging and is placed the animal management system of proprietary indoor design.This system introduces warm water heating system, and in 37 ℃, and the hot optical monitoring system of four-way is used for the skin temperature of monitor animal, the wall temperature of ambient temperature and device with temperature maintenance.For imaging, use 4% isoflurane induced anesthesia, remaining gas is made of 66% oxygen and the mixing of 30% nitrous oxide.Use 1.5% isoflurane under the gas condition of described similar oxygen and nitrous oxide, to obtain keeping of anesthesia.The animal of anesthesia placed cylindric variable radiation frequency resonance antenna inside (the birdcage resonance body amasss the molded lines circle) and be tuned to the mid frequency (resonant frequency when hydrone is accepted 7 tesla field intensity) of about 300MHz.Used imaging experiment scheme is T1 Weighted T urbo RARE (obtaining quick enhancing fast) the three-dimensional imaging sequence of carrying out on 7T BrukerBioSpin (Germany/USA) imaging control station.Used imaging parameters is: T1 Weighted T urbo-RARE 3D (3-dimension), and TE 13.3ms, TR 229.5, and Flipback on has the visual field of 8.0/3.5/3.5cm and 4 echoes of 256 * 256 * 256 matrixes.After obtaining baseline image, remain secured to animal in the animal support and use the 27G pin to the tail vein of animal, to come the whole body administration by intravenous injection

(the saline pH=7.4 with the 1x phosphate-buffered is diluted to 400 μ l) or contain

Immunoliposome complex (TfRscFv-Lip-Mag) (cumulative volume 400 μ l) and begin the 3-D imaging sequence immediately.Use the imaging of two kinds of solution in the consecutive days.

Scanning electron microscopy (SEM) (SEM)

Prepare liposome at GUMC

The sample solution of contrast agent and the complete nano-complex that is made of the cancer target strand TfR albumen of the complex that covers liposome, TfRscFv-Lip-Mag is sent to NIST and stores under dark cold preservation.For each imaging moiety, with the fresh diluent of 1: 3 volume of deionized water preparation and with 5 μ L droplet micro-injections to the standard 200-order TEM grid that constitutes by 30-60nm Formvar and 15-20nm carbon.Before being loaded into microscopical vacuum chamber, make droplet air drying 5 minutes on grid.Use Hitachi S-4800 field emission microscope, to carry out imaging at NIST.Interesting especially is, and SEM is applied to the NDA imaging is upside and downside secondary electron detector [SE (U) and SE (L)]--and use the comparison of SEM--with interpolation emission electron (TE) detector of normal mode, equipment is changed into low-voltage STEM.

Scanning probe microscopy (SPM)

Prepare liposome at GUMC

The sample solution of contrast agent and complete nano-complex are sent to NIST and store under dark cold preservation.For each imaging moiety, with the fresh diluent of 1: 3 volume of deionized water preparation and with 5 μ l droplet micro-injections to the silicon matrix of ultrasonic waves for cleaning, use native oxide or poly-L lysine to be coated with this substrate.The Veeco MultiMode microscope that use has Nanoscope IV controller obtains the SPM imaging.Carry out percussion mode (Veeco RTESP cantilever, the topography of～320-360kHz and k～20-60N/m), imaging and use the magnetic force microscopy (Veeco MESP 68kHz) at the tip that magnetic covers mutually by having Z control with life pattern.Along with exposing the dynamic imaging that drying of isolating nano-particle and aggregation and surface energy " be separated ", solution evaporation is used for understanding solvent seasoning to the result of granule stability and to using the influence of the obtainable various SPM radiography mechanism of SPM system.

The result

The tumour-specific targeting of the part-liposome nano-complex by carrying reporter gene

In order to assess the selectivity targeting of TfRscFv-LipA nano-complex to primary tumor and metastatic tumor, use the original position metastasis model, clinical setting near method of approximation, end user PanCa cell line CaPan-1.In 56 days, in liver and spleen, produced transfer (Alisauskus, R. etc., Cancer Research 55:5743a-5748s (1995)) with having demonstrated in the CaPan-1 xenotransplantation tumor section surgery original position implantation nude mice.According to the tumor in situ of in female nude mouse, inducing CaPan-1 described in the method.After about 5 weeks, make animal euthanasia and postmortem seek tumor in pancreas and other organs.As shown in Figure 1A, tumor growth is tangible widely in whole pancreas.Identical tumor nodule in the liver among the 1A shown in the haircut presents intensive beta galactosidase expression among the 1B.The 1A=postmortem; Tissue after the dyeing of 1A=beta galactosidase.There is metastatic tumor in four the various organs in five mices, comprises spleen, liver, lung, the adrenal gland, even in barrier film.Repeat this experiment, obtain similar result.

In order to set up selectivity target tumor and metastatic tumor, before putting to death mice, to carry the TfRscFv-LipA complex intravenous injection of pSVb (LacZ) plasmid DNA that is used for the beta galactosidase expression to mice, in 24 hours three times (per injection 40 μ g plasmid DNA).Inject and whole five mices were put to death in back 60 hours and collect various organs, comprise liver, lung, spleen and barrier film detect metastatic tumor and the painted existence of tumour-specific.The fresh sample dyeing that will be cut into 1mm thickness with X-gal produces blue with the place at expressing gene.The cancer target ability and the high transfection efficiency (Figure 1B) of complex have been proved by the existence of the reporter gene in the various organs of this animal.At liver, lung in adrenal gland and the barrier film, has clearly illustrated reporter gene only in the expression of metastatic tumor camber, although significantly not blue in normal adjacent tissue.Visible metastatic tumor (arrow) is the identical tumor nodule (arrow) of strong expression beta galactosidase among Figure 1B in Figure 1A liver, has confirmed the tumour-specific character of this nano-complex.In some mices, growth of tumor has also caused the tumor of the whole initial notch portion that is used to implant outstanding in the pancreas.In Figure 1B, also shown the Subcutaneous tumor of this strong blue dyeing that centers on by normal non-dyeing skin, shown tumor cell specific once more.Observe similar result in remaining mice and multiple experiment, therefore, the nano-complex of this whole body administration no matter they are where to produce in vivo, can both transmit plasmid DNA with targeting constitutional and metastatic tumor cell effectively.We wish that the potential of expanding this transfer system comprises contrast agent.This ability will cause the imaging and the cancer detection that improve.

Use the TfRscFv-Lip complex to transmit

In vitro study

Because

Be one of contrast agent the most frequently used in clinical, therefore select to be used for these researchs.In these initial experiments, detect complex and whether can use

The preparation and do like this and whether can improve the MRI signal.Because trypsin treatment will cause film to damage and contrast agent spills, therefore in these researchs, do not use attached cell from cell.Alternately be, used human lymphocyte leukaemia system, K562, it is grown as suspension culture.In addition, will remove in the precellular soft precipitation of imaging and washing any excessive

Or complex, make and only detect the signal relevant with cell.

1. the time dependence imaging by the TfRscFv-Lip-Mag nano-complex improves

Detected TfRscFv-Lip-

The Best Times of nano-complex transfection.Suggestion

Clinical dosage is 0.1mMole/kg.In these initial researchs, used the complex dosage (mice is proofreaied and correct people's less body weight and blood volume) of per 250 μ l transfection solution 0.3mMole/kg.With 20 to 90 minutes time of K562 cell transfecting.Based on imaging intensity, showed low-down transfection activity in 20 minutes.Yet, as shown in Fig. 2 A, in the time of 60 minutes, demonstrate a large amount of raisings of intensity with the complex cells transfected, compare with untreated cell.The intensity of untreated cell (202 ± 48) and empty labelling pipe (194 ± 43) do not have a significant difference, show that cell self does not cause detected signal.More importantly, plateau has appearred in transfection efficiency in the time of about 60 minutes, because cell transfecting 60 is identical (respectively doing for oneself 317 ± 46 and 317 ± 47) with 90 minutes relative intensity.

2.

The dose dependent imaging improves

Use 60 minutes as the transfection time, measured then and increase progressively content

Influence to TfRscFv-Lip-Mag complex imaging raising.The dosage of test is 0.05,0.3 and 0.9mMole/kg.Size and blood volume to mice are proofreaied and correct, and use in the complex of per 250 μ l transfection solution

Volume is 0.25 μ l, 1.5 μ l and 4.5 μ l.As shown in Fig. 2 B and table 1, as the function of the contrast agent content that comprises in the complex, image intensity improves and T1 shortens relaxation time.

Table 1: in the immunoliposome complex

The relative intensity of function and T1 relaxation time

Contrast agent dose (mM/kg)	Relative intensity	T1 (second)
			0.05(0.25μl)	293±50	1.43±0.007
0.3(1.5μl)	379±43	1.16±0.004
			0.9(4.5μl)	454±51	1.01±0.004

3. with free

The imaging of TfRscFv-Lip-Mag of comparing improves

Based on above-mentioned experiment, shown the TfRscFv-liposome can with

Compound and be sent to cell and be used for the figure image intensifying.In order to measure compound contrast agent enhanced level of comparing with independent reagent and the signal that proof is obtained is not owing to introduce

Exist caused, with free

Or the TfRscFv-Lip-Mag nano-complex is handled the K562 cell.For two kinds of solution, use the contrast agent (0.3 μ M/kg or 1.5 μ l/250 μ l transfection volumes) and the transfection time (60 minutes) of same amount.Although it is as expected, free

Demonstrate the contrast of raising with respect to untreated cell, the cell of handling with the TfRscFv-Lip-Mag complex has proved with untreated and free

The compare bigger raising of image intensity and shortened Ti relaxation time (Fig. 2 C, table 2) of the cell of handling.These results have not only proved the contrast agent absorption efficient that improves by the targeted nano complex, and show that viewed signal is not by not compound probably Cause.

Table 2: free compound with immunoliposome

Between relative intensity and the comparison of T1 relaxation time

Use the in-vivo image of TfRscFv-Lip-Mag to strengthen

The above external imaging tumor cell ratio of nano-complex of having determined

More effective separately.Yet in order to have the possibility of clinical use, complex must present similar effect in vivo.Identical human pancreas cancer original position mouse model (CaPan-1) is used for these research, with the above the same tumour-specific targeting that proves the complex that carries reporter gene.In addition, also used second kind of tumor model, subcutaneous prostate xenotransplantation mouse model (DU145).On 7T Bruker NMR, make the mice imaging that has CaPan-1 or DU145 tumor according to " method " part is described.In case place coil, use T1 Weighted T urbo RARE (obtaining quick enhancing fast) three-dimensional imaging sequence to obtain baseline image.In order to promote the image comparison, after baseline obtains, animal is maintained in the animal support, simultaneously by intravenous injection administration imaging solution.Commencing signal obtains in three minutes injecting.Deliver medicine to mice free (with clinical in carry out the same) or be included in the complex

Content is 10 μ L.This content equals twice used in 0.2mM/kg or the human body.Select this content to be because the 0.1mM/Kg of standard

People's dosage has obtained the signal of non-constant in mice separately.Dissociated at successive two days

Imaging with the TfRscFv-Lip-Mag complex.Also before the administration nano-complex, carry out baseline scan and confirmed owning of the previous day

Discharge.MR technology between two groups of images is consistent with window, regulates window and proofreaies and correct with the automatization's window feature for scanner.

Shown among Fig. 3 A-I in three independent mices

And the image of nano-complex-Mag.Fig. 3 A, among 3D and the 3G, surgery was implanted behind the CaPan-1 tumor cell four months, and animal is carried big tumor in situ.Compare with independent contrast agent, the resolution of raising and signal intensity are very obvious.In having second kind of mice of CaPan-1 tumor, observe similar result, be shown in Fig. 3 B, among 3E and the 3H.Have only bimestrial this animal to have tangible Subcutaneous tumor growth after the operation at whole cutting part.Also be checked through little abnormal mass by palpation.It is compound to be not only administration The signal of back Subcutaneous tumor is stronger, and in this scanning, demonstrating little tumor in situ (arrow) is significantly, and accepts

Animal in but be not like this.Similarly, with free Compare, behind the injection TfRscFv-Lip-Mag complex, definition that improves in the subcutaneous DU145 tumor and contrast are tangible (Fig. 3 C, 3F and 3I).At Fig. 3 B of expression two different tumor model cancer of pancreas (CaPan-1) and carcinoma of prostate (DU145), 3E and 3H and 3C rebuild on the image among 3F and the 3I with quantitative.In two kinds of situations, as expected, free

Exist and surpass the intensity (pixel) that baseline increases.Yet preparation causes the almost triple again enhancing of signal intensity in these two tumor models by the transmission of cancer target nano-complex.Therefore these studies have shown that and have incited somebody to action

When mixing in the TfRscFv-liposome complex, have the tumor visuality that improves in vivo in the situation, they show that this lesion detection approach of further research and development is used for the potential use of clinical use.

The physics characterization research

Although in vitro study has given compound

Be wrapped in the intravital evidence of lipid, and complicated microscopy (SEM and SPM) verified this and the more feature (for example, complex size) of TfRscFv-Lip-Mag.

1. do not have

The imaging of liposome

High-resolution imaging means the narrow depth of field, and therefore needs relative thin and flat sample.Thin degree changes according to technology, but surface and substrate effect--surface energy and symmetry reduce--have been dominated the typical structural capacity of biomaterial usually.This is especially true under the situation of liposome, has given their very thin characteristic (Foo, J.J etc., Annals of BiomedicalEngineering 31:1279-1286 (2003)).Therefore understanding the reliable method that is used to prepare with characterizing the two peacekeeping mechanical stabilities of separating liposome is an essential step.The purpose of this sign is to carry out the direct detection of the mechanical hardness and the magnetic characteristic of nano-particle, is not externally to link to each other simply with liposome to determine whether contrast agent is contained in the nano-particle veritably.

Approaching by SPM imaging surface landform and cantilever resonant frequency with percussion mode vibrating tip and connected cantilever.Feedback circuit is kept the vibration of cantilever with constant amplitude.Provide this constant amplitude with setting value, it is slightly smaller than the cantilever that freely swings.Because SPM is most advanced and sophisticated by various little power and surface interaction, cantilever excite and the surface on have phase shift between the replying of set point.For uneven surfaces, tip-surface interaction will be according to for example surface charge, and precipitous landform changes and mechanical hardness changes and changes.By changing setting value and observe specific feature and how to reply more soft or harder knocking, we can be associated this with replying that ad hoc structure such as liposome are expected.(amplitude signal is approximately 1.78V freely).The sequence of the SPM phasor of a pair of separation liposome that does not have a payload that has illustrated among Fig. 4 A-C.Fig. 4 A be the setting value imaging of 1.68V and substrate and liposome between the respective negative facial difference represent that tip-sample interacts and attract for liposome, provide with the values mutually of-3.5 degree.In the situation of suction phase mutual effect and negative, the phasor of liposome demonstrates dark-coloured, except ring crucial on the landform at liposome edge.Fig. 4 B has proved the effect of setting value being reduced to 1.45V: it is light tone that liposome demonstrates now, repelled because tip-sample interacts to become, and in this case, differing between liposome and the substrate is+8 degree.At last, Fig. 4 C demonstrates the further raising that differs at the setting value record of 1.35V, becomes+35 degree.

2. liposome

Imaging

Fig. 5 A-C represents the SPM and the SEM image of Magnevist (Lip+Mag) nano-particle of isolating liposome.The particulate distribution of sizes of single Lip+Mag is the 100-200nm diameter and shows that according to the classification of optical detecting to be enclosed with the liposome of imitating load approximately big by 50% than globular independent liposome.

The SPM landform that shows among Fig. 5 A shows and contains

Liposome after drying, have bimodal surface configuration, more complicated than the (not shown) of the simple oval surface of the liposome that does not contain payload.The SPM phase behavior is significantly different with the liposome of no payload, and outer shroud repels with respect to the center, has shown corresponding SPM phasor among Fig. 5 B.The interactional part of tip-sample that attracts and repel occurs in medium setting value.1.6 SPM phasor and the dependency between the SEM figure in the TE pattern that the setting value place obtains are tangible in Fig. 5 B and 5C.Liposome demonstrates uniform brightness (not shown) in whole particle among the SEM figure, and is similar by the even phasor that SPM obtains to us.Most advanced and sophisticated and cantilever changes with purposes in time.In addition, confirm that importantly the image that produces is not subjected to because the most advanced and sophisticated instable influence that the exogenous material on the tip causes.Therefore, with they frequent changes.Because each cantilever is slightly different for its resonance characteristics, so used setting value difference in the Figure 4 and 5.

3.TfRscFv-Lip-Mag the imaging of nano-complex

According to the complete TfRscFv-Lip-Mag nano-complex of the preparation described in the method and by SEM and SPM imaging.Result shown in Fig. 6 A and the 6B shows that solvent membrane has stood to be separated; Yet, can on exsiccant film, easily observe the example that separates NDS.Notice that the SEM bundle clearly causes some damages of film, but can be before the bundle damage becomes obviously several times with the granule multiple scanning.The outward appearance of complete complex is different from the situation of having only (Lip+Mag).Shape is rule not too, and the significant systematism of dry back surface of liposome is consistent with protein denaturation.In addition, SEM TE image shows between the outer shroud of liposome and the center boundary clearly, and (Lip+Mag) granule see not too obvious, and change of shape is bigger.It is consistent that this existence with the liposome internal protein has changed the viewpoint of the infiltration outflow thing that passes liposome in the film dry run.

Use the magnetic force micro-imaging ability of SPM (MFM) can obtain particulate other information about these NDS.Because contain gadolinium

Magnetic moment very big, should use magnetized SPM most advanced and sophisticated come with liposome in spissated orientation

Interact.MFM for several about 10-200nm diameter nano-particle is shown among Fig. 7 A and the 7B.Use the rising mode capabilities of SPM, determined that the image that produces is magnetic in itself really.In this pattern, obtained the topography under the normal percussion mode condition.Use the reference table surface information with the certain height of tip offset then, then at the height scanning of a surface of this raising away from the surface.This has removed the influence of landform to signal.By magnetic phase diagram looked like to provide leave surperficial 15nm or more high altitude sentence the MFM image that lift mode obtains.The appearance of signal has confirmed the existence of the gadolinium of parcel in the complex.Fig. 7 A is the SPM landform/magnetic phase diagram picture of complete TfRscFv-Lip-Mag nano-complex.Appearance by the quadripole increment signal that attracts and repel the plane magnetic interaction to constitute among Fig. 7 B shows that this interactional inducement is in the NDS

Inhomogeneous annular spread, consistent with SEM and non-magnetic SPM phasor.

Discuss

Result described herein has proved that we can wrap up MR preparation commonly used

And with it external or be sent to tumor cell in the animal model in position, and do like this and produced than with not compound

More clear and the intensive image of seeing.

As shown in fig. 1, nano-complex of the present invention can the targeted metastatic disease, therefore improves the detection sensitivity of metastatic tumor.Use SEM and SPM, proved that the TfRscFv-liposome complex is at parcel

The time kept nano-scale (granule that has shown about 100-200nm among Fig. 6 and 7).Verified structure and the mechanical features that contains the liposome of payload significantly is different from those that do not contain, and therefore confirmed that liposome has wrapped up really

MFM imaging by complex has further confirmed this point.

Do not wish to be subjected to the constraint of following theory, (Lip+Mag) the trial interpretation of internal structure is that small ledge in the SPM topography is represented the PHASE SEPARATION that liposome limits, that is, intensive around the granule periphery

The formation of-lipid annular spread, preferential at the water at granule center.This replying may be because several important factors cause: at first, and according to manufacturer, The feature of solution is pH～6.5-8,4.9 viscosity when 1,960 Morie osmolarity and 20 ℃.

The specious chemical fundamentals of this solution separating that marks in the data sheet: meglumine salt is separated from complex fully, has therefore changed the local penetration condition.Add the charge interaction with gadolinium complex and cationic-liposome, these interactions can provide intensive driving force, the intravital height of lipid is oozed be separated.At stabilized liposome with when structural support in the solution is provided, cationic-liposome and

CHARGE DISTRIBUTION between the solution is effectively, and is tangible in blood flow.The structural support of this raising has important benefit for these researchs, because this allows most of granule to be kept perfectly in the film dry run, has only the observed large-scale decomposition of solution of liposome opposite with use.

Embodiment has before proved the successful parcel of mr angiography agent in the immunoliposome complex of the present invention.By conventional transmission of the surpassing of complex proof

The figure image intensifying show that this system has improved the ability by the cancer earlier detection of MRI.

Embodiment 2

Imaging in the different cell lines relatively

Fig. 8 A-8H has shown the raising MR imaging of using part-HK-liposome-Mag nano-complex in two various cancers models.Use same ratio and the method shown in the embodiment 1 to prepare nano-particle used among this embodiment.Lower back portion subcutaneous injection people breast carcinoma MDA-MB-435 (Fig. 8 E-8H) or PC-3 (DU145) (Fig. 8 A-8D) at female athymic mouse.With free

Or TfRscFv-liposome nano-complex (scLip-Mag), or comprise HoKc peptide and same dose

TfRscFv-HK-liposome nano-complex (scLip-HK-Mag) continuously respectively intravenous injection in three days (by the tail vein) be injected in three mices.

This content be the twice that equals to deliver medicine to people's patient dose.The overall solution volume of administration is 400 μ l in all situations.Just before two kinds of nano-complexes of administration, carry out baseline scan and confirm owning from the previous day

Discharge.MR technology and window between four groups of images are constant, regulate window and proofread and correct with the automatization's window feature to scanner.Figure demonstrates the difference of MRI signal in the mice that has Subcutaneous tumor, wherein definition that injection improves behind the scLip-Mag in tumor of prostate (DU145) (Fig. 8 A-8D) and breast tumor (435) (Fig. 8 E-8H) and contrast are significantly, behind the injection scLip-HK-Mag even more obvious.

Fig. 9 A-9C has shown that CaPan-1 Subcutaneous tumor and original position metastatic tumor model pass through the tumour-specific targeting of TfRscFv-HK-liposome-Mag nano-complex.According in female athymic mouse, inducing subcutaneous CaPan-1 xenotransplantation tumor described in the method for embodiment 1.Collecting tumor also will

In the individual cells suspension be injected in the pancreas of surgical exposure.In injection eight weeks of back, will carry

The TfRscFv-liposome complex that contains or do not contain HoKC (HK) peptide be injected in the mice in continuous two days.Giving the cumulative volume of drug solns in all situations is 400 μ l.Just before the administration nano-complex, carry out baseline scan and confirm owning from the previous day

Discharge.MR technology and window between three groups of images are constant, regulate window and proofread and correct with the automatization's window feature to scanner.Similar to Fig. 8 A-8H, observe the imaging resolution that Subcutaneous tumor (white arrow) and metastatic lesions improve, as shown in table 3.

Table 3: free and compound The intensity that exceeds baseline increases

Embodiment 3

The comparison of the Dynamic MRI scanning of subcutaneous PANC-1 tumor behind systemic injection free (not compound) or the TfRScFv-Lip-Magnevist

Carrying out following experiment comes the comparison whole body to transmit the absorption between free (not compound) and TfRscFv-Lip-Mag and the speed and the level of discharge in the tumor of back.Be suspended in Matrigel by injection ^TMIn the collagen basement membrane matrix (BD Biosciences) 2 * 10 ⁷The PANC-1 cell is induced the subcutaneous xenotransplantation tumor of PANC-1 in female athymic mouse.Approximately 2.5-3 is used for imaging with animal after week.Prepare cationic-liposome (DOTAP:DOPE) (referring to U.S. publication application No.2003/0044407 according to described before by the ethanol injection method; Xu L etc., Molecular Cancer Therapeutics 1:337-346 (2002) is hereby incorporated by its disclosure).Used targeting moiety is an anti-TfR single chain antibody fragments (TfRscFv) in these researchs.

In order to wrap up preparation, TfRscFv and liposome mixed in certain proportion being incorporated in incubated at room 1-30 minute, 5-20 minute suitably, optimum 10-12 minute.Will

Add in this solution, mix and once more at incubated at room 1-30 minute, 5-20 minute suitably, more suitably 10-12 minute.When in preparation is used for body, using, add sucrose or glucose final concentration, suitably 1-20% to 0.5-50%, being 10% for sucrose most suitably, is 5% for glucose, and at incubated at room 1-30 minute, 5-25 minute suitably, optimum 15-20 minute.Use above-mentioned method 1mg preparation than 0.33-1.17 μ gTfRscFv than the ratio of 10-35 μ g liposome (suitably the 1mg preparation than 0.5-1.0 μ gTfRscFv than 14-28 μ g liposome, optimum 1mg preparation than 0.71 μ g TfRscFv than 21 μ g liposomees) form complex.Acceptable complex size range is about 20 to 1000nm, and about suitably 50 to 700nm, and optimum about 100 to 500nm.Use 4.7mgMagnevist at this, 99 μ g liposomees and 3.3 μ g TfRscFv, use final concentration are 5% glucose formation complex.

With as above the inductive PANC-1 of having Subcutaneous tumor mouse anesthesia and place the animal mounting system.Use 4% isoflurane induced anesthesia, residual gas comprises 66% oxygen and 30% nitrous oxide mixture.Under the gas condition of described similar oxygen and nitrous oxide, use 1.0 to 2.0% (preferred 1.5%) isoflurane to obtain keeping of anesthesia.The animal of anesthesia placed cylindric variable radiation frequency resonance antenna inside (the birdcage resonance body amasss the molded lines circle) and be tuned to the mid frequency (resonant frequency when hydrone is accepted 7 tesla field intensity) of about 300MHz.Used imaging experiment scheme is T1 Weighted T urbo RARE (obtaining quick enhancing fast) the two-dimensional imaging sequence of carrying out on 7T Bruker BioSpin (Germany/USA) imaging control station.Used imaging parameters is: T1 weighting 2D (2-dimension), and TE 10.21ms, TR 420.3, and Flipbackoff has 8 echoes of 5.12/5.12cm visual field.After obtaining baseline image, remain secured to animal in the animal support and use the 27G pin to come whole body administration dissociate (not compound) to the tail vein of animal by intravenous injection (Mag, or at this gad-d) or contain the TfRscFv-Lip-Mag (cumulative volume 50-1000 μ l, 100-500 μ l suitably, optimum 200-400 μ l) of same amount Mag and begin imaging sequence immediately.In intercycle ground multiple scanning in two hours (meansigma methods 2,1.3 minutes), and measure and draw pixel intensity.Identical mice is used for the imaging of free and complex.In successive natural law, carry out imaging, at first use free Mag.

As shown in Figure 10, compare, behind the intravenous injection complex, have the signal of remarkable higher level in the tumor with free preparation.More importantly, in the time course of experiment, kept this higher level.

Embodiment 4

Detect the hepatic metastases of CaPan-1 by TfRscFv-Lip-HoKC-Magnevist

Carry out following experiment and assess the ability that TfRscFv-Lip-HoKC-Mag complex of the present invention detected and improved the imaging of metastatic tumo(u)r.For example, detected metastatic tumor from cancer of pancreas, yet, use complex of the present invention and method can obtain imaging (for example, prostate, melanoma from the cancer metastasis tumor of any kind, kidney, mammary gland, stomach, liver, ovary, bladder, head and neck, brain, the entity tumor of bone and any other type).Be suspended in Matrigel by injection ^TMIn the collagen basement membrane matrix (BD Biosciences) 0.5 to 1 * 10 ⁷The CaPan-1 cell is induced the subcutaneous xenotransplantation tumor of CaPan-1 in female athymic mouse.After about eight weeks, collect tumor and prepare the single-cell suspension liquid of tumor.To be suspended in Matrigel equally according to described before ^TMIn 1.2-1.5 * 10 ⁷Injection cell (Alisauskus to the pancreas of female athymic mouse surgical exposure, R., G.Y., and Gold, D.V., Initial studies of monoclonal antibody PAM4 targeting toxnografted orthotopic pancreatic cancer (preliminary study of the monoclonal antibody PAM4 of the heteroplastic original position cancer of pancreas of targeting), Cancer Research 55,5743s-5748s (1995)).

Prepare cationic-liposome (DOTAP:DOPE) (referring to U.S. publication application No.2003/0044407 by described ethanol injection method before; Xu L etc., MolecularCancer Therapeutics 1:337-346 (2002) is incorporated herein by reference its disclosure).The HoKC peptide K[K (H) KKK] ₅-K (H) KKC} (SEQ ID NO:1) has terminal cysteine to allow to put together maleimide base group.Therefore, when using the HoKC peptide, Liposomal formulation also comprises 0.1 to 50 molar percentage of TL, more preferably the 1-10 molar percentage of TL, most preferably the N-maleimide of 5 molar percentages of TL-phenylbutyric acid salt-DOPE (MPB-DOPE).Prepare HoKC liposome (Yu as previously mentioned, W etc., Enhancedtransfection efficiency of a systemically deliveredtumor-targeting immunolipoplex by inclusion of a pH-sensitivehistidylated oligolysine peptide (by comprising the transfection efficiency of the cancer target immunity lipoplex that whole body that the few lysine peptide of pH sensitivity group acyl ammonia salt improves transmits), Nucleic Acids Research 32, e48 (2004)).Used targeting moiety is an anti-TfR single chain antibody fragments (TfRscFv) in these researchs.

In order to wrap up preparation, with TfRscFv and liposome with specific mixed and at incubated at room 1-30 minute, 5-20 minute suitably, most suitably 10-12 minute.Will

Add in this solution, mix and once more at incubated at room 1-30 minute, 5-20 minute suitably, optimum 10-12 minute.When in preparation is used for body, using, add sucrose or glucose final concentration, suitably 1-20% to 0.5-50%, being 10% for sucrose most suitably, is 5% for glucose, and at incubated at room 1-30 minute, 5-25 minute suitably, most suitably 15-20 minute.(than 14-28 μ g liposome-HoKC, optimum 1mg preparation forms complex than 0.71 μ g TfRscFv than 21 μ g liposomees-HoKC) to the 1mg preparation than 0.5-1.0 μ g TfRscFv suitably than the ratio of 10-35 μ g liposome-HoKC than 0.33-1.17 μ gTfRscFv to use above-mentioned method 1mg preparation.Acceptable complex size range is about 20 to 1000nm, and about suitably 50 to 700nm, and optimum about 100 to 500nm.Use 4.7mg Magnevist at this, 99 μ g liposomees and 3.3 μ gTfRscFv, use final concentration are 5% glucose formation complex.

The animal mounting system is anaesthetized and placed to the mice (approximately 12 weeks behind the surgical operation implantation tumour cell) that will have above inductive CaPan-1 tumor in situ.Use 4% isoflurane induced anesthesia, residual gas comprises 66% oxygen and 30% nitrous oxide mixture.Under the gas condition of described similar oxygen and nitrous oxide, use 1.0 to 2.0% (preferred 1.5%) isoflurane to obtain keeping of anesthesia.The animal of anesthesia placed cylindric variable radiation frequency resonance antenna inside (the birdcage resonance body amasss the molded lines circle) and be tuned to the mid frequency (resonant frequency when hydrone is accepted 7 tesla field intensity) of about 300MHz.Used imaging experiment scheme is T1 Weighted T urbo RARE (obtaining quick enhancing fast) the three-dimensional imaging sequence of carrying out on 7T BrukerBioSpin (Germany/USA) imaging control station.Used imaging parameters is: T1 Weighted T urbo-RARE 3D (3-dimension), and TE13.3ms, TR229.5ms, Flipback on has 4 echoes of 8.0/3.5/3.5cm visual field and 256 * 256 * 256 matrixes.After obtaining baseline image, remain secured to animal in the animal support and use the 27G pin to the tail vein of animal, to come whole body administration TfRscFv-Lip-HoKC-Mag (cumulative volume 50-1000 μ l by intravenous injection, 100-500 μ l suitably, optimum 200-400 μ l) and begin the 3D imaging sequence immediately.

After the imaging, with animal euthanasia and estimate the existence of metastatic tumor.Use method known to a person of ordinary skill in the art to take out liver, be fixed in the formalin, paraffin embedding, section is also used H﹠amp; E dyeing.By microscopy section and take observed metastatic tumor.

Figure 11 A-11A: Figure 11 A: before the radiography.Figure 11 B:TfRcFv-Lip-HoKC-Mag injection, Figure 11 C: histology.The original position cancer of pancreas has shown the raising (short white arrow) of using TfRcFv-Lip-HoKC-Mag.Be connected two zones representing than the white arrow in the section of back and the tumor of representing initial original position with short.With with primary tumor in the identical pattern seen observe little metastatic tumor (thick white arrow) and improve.The thin expansion of liver (long thin arrow) be positioned at metastatic tumor near.Postmortem (not shown) and histology (image right) have confirmed exist (black arrow) of metastatic tumor, and metastatic tumor directly approaches expansion in abutting connection with the length of liver.Notice the similarity of the shape and the outward appearance on the MRI of one of metastatic tumo(u)r thin slice.

Embodiment 5

The pulmonary metastases of the raising by TfRscFv-Lip-Magnevist detects

Carrying out following experiment proves and compares when not using complex administration preparation that intravenous administration is (or by any other suitable way, for example, but be not limited to IT, ID, IM can strengthen the detection of metastatic tumor when IP) the present invention carries the complex of preparation.By intravenous injection 1 to 10 * 10 ⁴The RenCa cell is induced lung tumor in female Balb/C mice.Therefore this method causes almost staying in specially the metastatic tumor in the animal lung, and will cause as primary disease or as the animal model system of the cancer of the lung tumor of metastatic tumor as any.Approximately 2-4 is used for imaging with animal after week.

Prepare cationic-liposome (DOTAP:DOPE) (referring to U.S. publication application No.2003/0044407 according to described before by the ethanol injection method; XuL etc., Molecular Cancer Therapeutics 1:337-346 (2002) is hereby incorporated by its disclosure).Used targeting moiety is an anti-TfR single chain antibody fragments (TfRscFv) in these researchs.

In order to wrap up preparation, with TfRscFv and liposome with specific mixed and at incubated at room 1-30 minute, 5-20 minute suitably, most suitably 10-12 minute.Will

Add in this solution, mix and once more at incubated at room 1-30 minute, 5-20 minute suitably, optimum 10-12 minute.When in preparation is used for body, using, add sucrose or glucose final concentration, suitably 1-20% to 0.5-50%, being 10% for sucrose most suitably, is 5% for glucose, and at incubated at room 1-30 minute, 5-25 minute suitably, most suitably 15-20 minute.Use above-mentioned method 1mg preparation than 0.33-1.17 μ gTfRscFv than the ratio of 10-35 μ g liposome (suitably the 1mg preparation than 0.5 to 1.0 μ g TfRscFv than 14-28 μ g liposome, optimum 1mg preparation than 0.71 μ gTfRscFv than 21 μ g liposomees) form complex.Acceptable complex size range is about 20 to 1000nm, and about suitably 50 to 700nm, and optimum about 100 to 500nm.Use 4.7mg Magnevist at this, 99 μ g liposomees and 3.3 μ g TfRscFv, use final concentration are 5% glucose formation complex.

To have the mouse anesthesia of above inductive tumor and place the animal mounting system.Use 4% isoflurane induced anesthesia, residual gas comprises 66% oxygen and 30% nitrous oxide mixture.Under the gas condition of described similar oxygen and nitrous oxide, use 1.0 to 2.0% (preferred 1.5%) isoflurane to obtain keeping of anesthesia.The animal of anesthesia placed cylindric variable radiation frequency resonance antenna inside (the birdcage resonance body amasss the molded lines circle) and be tuned to the mid frequency (resonant frequency when hydrone is accepted 7 tesla field intensity) of about 300MHz.Used imaging experiment scheme is the T1 weighting two dimension Turbo Multislice-Multiecho imaging sequence that carries out on 7T Bruker BioSpin (Germany/USA) imaging control station.Used imaging parameters is: T1 weighting 2D (2-dimension), and TE 10.21ms, TR400ms, Flipback off has 8 meansigma methodss of 3.84 * 3.84cm visual field and 256 * 256 matrixes.After obtaining baseline image, remain secured to animal in the animal support and use the 27G pin to come whole body administration dissociate (not compound) to the tail vein of animal by intravenous injection

(gad-d) or contain the TfRscFv-Lip-Mag (cumulative volume 50-1000 μ l, 100-500 μ l suitably, optimum 200-400 μ l) of same amount Mag and begin imaging sequence immediately.The pixel intensity of measurement and drawing image.Identical mice is used for the imaging of free and complex.In successive natural law, carry out imaging.

As shown in Figure 12 A-12E, compare with free preparation, behind the intravenous injection complex, there is the signal of remarkable higher level in the tumor.Therefore, compare with the method for the free preparation of the administration of using always, complex of the present invention has also improved the detection of big relatively metastatic tumor in the pulmonary.

Embodiment 6

The detection of the little pulmonary's metastatic tumor that improves by TfRscFv-Lip-Magnevist

Carry out following experiment and prove that intravenous administration (or by any other suitable way, for example, but is not limited to, IT, ID, IM, metastatic tumor that can not be detected very little in the time of can detecting administration when IP) the present invention has the complex of preparation and do not use the preparation of complex.By intravenous injection 1 to 10 * 10 ⁴The RenCa cell is induced lung tumor in female Balb/C mice.Therefore this method causes almost staying in specially the metastatic tumor in the animal lung, and will cause as primary disease or as the animal model system of the cancer of the lung tumor of metastatic tumor as any.Approximately after 7-9 days, animal is used for imaging.

Prepare cationic-liposome (DOTAP:DOPE) (referring to U.S. publication application No.2003/0044407 according to described before by the ethanol injection method; XuL etc., Molecular Cancer Therapeutics 1:337-346 (2002) is hereby incorporated by its disclosure).Used targeting moiety is an anti-TfR single chain antibody fragments (TfRscFv) in these researchs.

In order to wrap up preparation, with TfRscFv and liposome with specific mixed and at incubated at room 1-30 minute, 5-20 minute suitably, most suitably 10-12 minute.Will Add in this solution, mix and once more at incubated at room 1-30 minute, 5-20 minute suitably, optimum 10-12 minute.When in preparation is used for body, using, add sucrose or glucose final concentration, suitably 1-20% to 0.5-50%, being 10% for sucrose most suitably, is 5% for glucose, and at incubated at room 1-30 minute, 5-25 minute suitably, most suitably 15-20 minute.Use above-mentioned method with the 1mg preparation than 0.33-1.17 μ gTfRscFv than the ratio of 10-35 μ g liposome (suitably the 1mg preparation than 0.5 to 1.0 μ g TfRscFv than 14-28 μ g liposome, optimum 1mg preparation than 0.71 μ gTfRscFv than 21 μ g liposomees) form complex.Acceptable complex size range is about 20 to 1000nm, and about suitably 50 to 700nm, and optimum about 100 to 500nm.Use 4.7mg Magnevist at this, 99 μ g liposomees and 3.3 μ g TfRscFv, use final concentration are 5% glucose formation complex.

To have the mouse anesthesia of above inductive tumor and place the animal mounting system.Use 4% isoflurane induced anesthesia, residual gas comprises 66% oxygen and 30% nitrous oxide mixture.Under the gas condition of described similar oxygen and nitrous oxide, use 1.0 to 2.0% (preferred 1.5%) isoflurane to obtain keeping of anesthesia.The animal of anesthesia placed cylindric variable radiation frequency resonance antenna inside (the birdcage resonance body amasss the molded lines circle) and be tuned to the mid frequency (resonant frequency when hydrone is accepted 7 tesla field intensity) of about 300MHz.Used imaging experiment scheme is the T1 weighting two dimension Turbo Multislice-Multiecho imaging sequence that carries out on 7T Bruker BioSpin (Germany/USA) imaging control station.Used imaging parameters is: T1 weighting 2D (2-dimension) imaging sequence, and TE 10.21ms, TR572.99ms, Flipback off has 8 meansigma methodss of 2.56 * 2.56cm visual field and 256 * 256 matrixes.After obtaining baseline image, remain secured to animal in the animal support and use the 27G pin to come whole body administration dissociate (not compound) to the tail vein of animal by intravenous injection

(gad-d) or contain the TfRscFv-Lip-Mag complex (cumulative volume 50-1000 μ l, 100-500 μ l suitably, optimum 200-400 μ l) of same amount Mag and begin imaging sequence immediately.The pixel intensity of measurement and drawing image.Identical mice is used for the imaging of free and complex.In successive natural law, carry out imaging.Under this visual field, 5 pixels equal people's tumor of about 3mm of detecting by CT.

As shown in Figure 13 A-13D, behind the injection complex but be not the metastatic tumor (following arrow) that can detect 4 pixels behind the free gad-d (corresponding to the about metastatic tumor of diameter 0.4mm).In addition, compare with free gad-d, signal significantly improves in second a little bigger slightly metastatic tumor (above arrow).Therefore, compare with the method for the free preparation of the administration of using always, complex of the present invention has also improved the detection of the medium and small metastatic tumor of pulmonary.Use complex of the present invention also to detect the littler metastatic tumor (equaling the tumor of about diameter 0.3mm) of about 3 pixels, but be to use free gad-d not detect (Figure 14 A-14D).

Use and top identical tumor model system, behind the intravenous injection complex of the present invention, can detect even the tumor of smaller szie.Imaging parameters also is T1-weighting 2D (2-dimension) Mutltislice-Multiecho imaging sequence as used herein, TE10.21ms, TR=630.8ms, Flipback off, 8 meansigma methodss of 2.56 * 2.56cm visual field and 256 * 256 matrixes.As shown in Figure 15 A-15B, can detect the tuberosity of 1-2 pixel by complex.When scanning, MRI observes nodule N 1 and N2.Because their very little (1-2 pixels) so that can not determine in fact whether they provide signal above background are used Image J software measurement intensity, and are measured two nodular minimums, maximum, meansigma methods and standard deviation (SD).Statistically, not noise but real if, there is 95% confidence level tuberosity greater than the 2SD on the maximum+basis on basis in nodular maximum.Tuberosity 2 is obviously in this 95% confidence level, and tuberosity 1 is just in the limit, and therefore the enhancing by complex also is real tumor mass probably.After the imaging, use standardization program known to a person of ordinary skill in the art to take out the lung of this animal, be fixed in formalin, paraffin embedding, section is also used H﹠amp; E dyeing.By the microscopy section, and take observed metastatic tumor.As Figure 16 (small-power, 2X) and Figure 17 (high-power, shown in 10X),, find two metastatic tumors of about 0.1mm size in the identical lobe of the lung and general position as desired based on MRI.Equal in distance between two tuberositys of measurement and discovery on the MRI image (～600nm) measured based on the histology.Therefore, in fact the tumor mass of these very little Histological determinings represents to use complex of the present invention according to the detected tuberosity of MRI.Find to be higher than usually with CT at this and see that CT is the common method that detects the lung primary tumor and be derived from the pulmonary metastases of other cancer types for the level of sensitivity that pulmonary metastases detects.Obviously this has represented unexpected and surprising result.

Embodiment 7

Detect subpleural pulmonary metastases by TfRscFv-Lip-Magnevist

Use among the above embodiment 6 described identical tumor model system of Figure 13 and 14 and imaging parameters, can also detect the metastatic tumor under the visceral pleura shown in Figure 18 A-18F.This is unexpected and surprising, because use the MR I imaging agents that in fact can not enter the non-complex reagent of cell at present can not detect the metastatic tumor of this position.This provides important advantage for the cancer of earlier detection and treatment pulmonary carcinoma and other types.

Embodiment 8

The detection that the melanoma lung that improves by TfRscFv-Lip-Magnevist shifts

About the detection/treatment of metastatic tumor of pleura, clinic control is very difficult to realize, and the measurement of helpfulness also is difficult.The result who presents in this embodiment shows that complex of the present invention can arrive and therefore the transfection metastatic tumor of pleura also also can be used for treating them.In addition, complex of the present invention can be the imaging tool that is used for measuring the effectiveness of this or any other treatment.

Carry out following experiment prove intravenous (or by any other suitable way, such as but not limited to IT, ID, IM can detect those metastatic tumors that are not limited to from renal cell carcinoma when IP) administration the present invention has the complex of preparation.By intravenous injection 0.1 to 5 * 10 ⁵The B16/F10 mouse black-in tumor cell is induced lung tumor in female C57/B16 mice.Therefore this method causes almost staying in specially the metastatic tumor in the animal lung, and will cause as primary disease or as the animal model system of the cancer of the lung tumor of metastatic tumor as any.Approximately 2-4 is used for imaging with animal after week.

Prepare cationic-liposome (DOTAP:D0PE) (referring to U.S. publication application No.2003/0044407 according to described before by the ethanol injection method; XuL etc., Molecular Cancer Therapeutics 1:337-346 (2002) is hereby incorporated by its disclosure).Used targeting moiety is an anti-TfR single-chain antibody part (TfRscFv) in these researchs.

In order to wrap up preparation, with TfRscFv and liposome with specific mixed and at incubated at room 1-30 minute, 5-20 minute suitably, most suitably 10-12 minute.Will Add in this solution, mix and once more at incubated at room 1-30 minute, 5-20 minute suitably, optimum 10-12 minute.When in preparation is used for body, using, add sucrose or glucose final concentration, suitably 1-20% to 0.5-50%, being 10% for sucrose most suitably, is 5% for glucose, and at incubated at room 1-30 minute, 5-25 minute suitably, most suitably 15-20 minute.Use said method with the 1mg preparation than 0.33-1.17 μ gTfRscFv than the ratio of 10-35 μ g liposome (suitably the 1mg preparation than 0.5 to 1.0 μ g TfRscFv than 14-28 μ g liposome, optimum 1mg preparation than 0.71 μ gTfRscFv than 21 μ g liposomees) the formation complex.Acceptable complex size range is about 20 to 1000nm, and about suitably 50 to 700nm, and optimum about 100 to 500nm.Use 4.7mg Magnevist at this, 99 μ g liposomees and 3.3 μ g TfRscFv, use final concentration are 5% glucose formation complex.

To have the mouse anesthesia of above inductive tumor and place the animal mounting system.Use 4% isoflurane induced anesthesia, residual gas comprises 66% oxygen and 30% nitrous oxide mixture.Under the gas condition of described similar oxygen and nitrous oxide, use 1.0 to 2.0% (preferred 1.5%) isoflurane to obtain keeping of anesthesia.The animal of anesthesia placed cylindric variable radiation frequency resonance antenna inside (the birdcage resonance body amasss the molded lines circle) and be tuned to the mid frequency (resonant frequency when hydrone is accepted 7 tesla field intensity) of about 300MHz.Used imaging experiment scheme is the T1 weighting two dimension Turbo Multislice-Multiecho imaging sequence that carries out on 7T Bruker BioSpin (Germany/USA) imaging control station.Used imaging parameters is: T1 Weighted T urbo-RARE 2D (2-dimension) imaging sequence, and TE 10.21ms, TR1418.13ms, Flipback off has 8 meansigma methodss of 3.84 * 3.84cm visual field and 256 * 256 matrixes.After obtaining baseline image, remain secured to animal in the animal support and use the 27G pin to come whole body administration dissociate (not compound) to the tail vein of animal by intravenous injection

(gad-d) or contain the TfRscFv-Lip-Mag complex (cumulative volume 50-1000 μ l, 100-500 μ l suitably, optimum 200-400 μ l) of same amount Mag and begin imaging sequence immediately.The pixel intensity of measurement image.Identical mice is used for the imaging of free and complex.In successive natural law, carry out imaging.

Shown in Figure 19 A-19B, inject compound

The back detects two little metastatic tumors (arrow) in lung.Graphical representation is by two different sections of lung.

Use and above the same tumor model system (B16/F10 melanoma) and imaging parameters, the dynamic curve in the use Image J software is measured in another animal behind the baseline, dissociates

Metastatic tumor pixel intensity behind back and the TfRscFv-Lip-Mag, and numerical value compared.Shown in following table 4, the maximum that complex demonstrates above baseline value strengthens.The difference that standard deviation demonstrates between complex and the baseline value is significant, and dissociates

And between the baseline then be not.

The comparison of the signal intensity in the table 4:B16/F10 pulmonary metastases

List of references

1.Gillies, RJ., etc., Neoplasia (New York) 2:139-451 (2000)

2.Degani, H., etc., Thrombosis﹠amp; Haemostasis ￡ 9:25-33 (2003)

3.Xu, L., etc., Human Gene Therapy 10:2941-2952 (1999)

4.Xu, L., etc., Tumor Targeting 4:92-104 (1999)

5.Xu, L., etc., Molecular Medicine 7:723-734 (2001)

6.Xu L, etc., Molecular Cancer Therapeutics 7:337-346 (2002)

7.Rait, A., etc., Molecular Medicine 5:476-487 (2002)

8.Rait, A., etc., Ann.NY.Acad.Sci.1002:1-12 (2003)

9.Cristiano, RJ., and Curiel, D.T., Cancer Gene Therapy5:49-57 (1996)

10.Cheng，P.W.，Human Gene Therapy 7：275-282(1996)

11.Keer, H.N., etc., Journal of Urology 743:381-385 (1990)

12.Rossi, M.C., and Zetter, B.R., Proc.Natl.Acad.Sci. (USA) 89:6197-6201 (1992)

13.Elliott, R.L., etc., Ann.NY.Acad.Sd.698:159-166 (1993)

14.Thorstensen, K., and Romslo, I., Scand.J.Clin.Lab.Investig. (Supp.) 275:113-120 (1993)

15.Miyamoto, T., etc., Intl.J.Oral Maxillofacial Surg.23:430-433 (1994)

16.Ponka, P. and Lok, C.N., Int ' l J.Biochem.Cell Biol.37:1111-1137 (1999)

17.Haynes, B.F., etc., J.Immunol 727:347-351 (1981)

18.Batra, J.K., etc., Molecular ﹠amp; Cellular Biology77:2200-2205 (1991)

19.Jain, R.K. and Baxter, L.T., Cancer Res.48:7022-1032 (1988)

20.Wolfert, M.A., etc., Human Gene Therapy 7:2123-2133 (1996)

21.Dunlap, D.D., etc., Nucleic Acids Research 25:3095-3101 (1997)

22.Kawaura, C, etc., FEBS Letters 421:69-72 (1998)

23.Choi, Y.H., etc., Human Gene Therapy 10:2657-2665 (1999)

24.Diebel, C.E., etc., Nature 406:299-302 (2000)

25.Rasa, M., etc., J.Coll.Interface Sci 250:303-315 (2002)

26.Yu, W., etc., Nucleic Acids Research, 32 (5): e48 (2004)

27.Alisauskus, R., etc., Cancer Research 55:5743s-5748s (1995)

28.Foo, J.J., etc., Annals of Biomedical Engineering 31:1279-1286 (2003)

29.Xu, L, etc., Human Gene Therapy 73:469-481 (2002)

30.Freedman, M., etc., SPIE Medical Imaging:Physiology andFunction from Multidimensional Images 4321:163-167 (2001)

31.Wisner, E.R., etc., Investigative Radiology 37:232-239 (2002)

32.Winter, P.M., etc., Circulation 108:2270-2274 (2003)

33.Morawski, A.M., etc., Magnetic Resonance in Medicine51:480～486 (2004)

All publications of mentioning in this description, patent and patent application are hereby incorporated by, and extremely as each independent publication, patent or patent application specially also represent to introduce the degree of reference separately.

Sequence table

<110>Georgetown University

Chang，Esther H.

Pirollo，Kathleen F.

＜120〉in order to improve the cancer target nano-delivery system of MRI earlier detection

<130>2474.006PC01

<150>US 60/728,303

<151>2005-10-20

<160>1

<170>PatentIn version 3.3

<210>1

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜223〉chemosynthesis-HoKC

<400>1

Lys Lys His Lys Lys Lys Lys His Lys Lys Lys Lys His Lys Lys Lys

1 5 10 15

Lys His Lys Lys Lys Lys His Lys Lys Lys Lys His Lys Lys Cys

20 25 30

Claims (48)

1. prepare the method for antibody or antibody fragment target cationic immunoliposome complex, comprising:

(a) preparation antibody or antibody fragment;

(b) described antibody or antibody fragment are mixed with cationic-liposome form the cation immunoliposome, wherein said antibody or antibody fragment are chemically being puted together with described cationic-liposome; With

(c) described cation immunoliposome is mixed with preparation form described antibody or antibody fragment target cationic immunoliposome complex.

2. the process of claim 1 wherein antibody is mixed with described cationic-liposome.

3. the process of claim 1 wherein antibody fragment is mixed with described cationic-liposome.

4. the method for claim 3, wherein said antibody fragment is a strand Fv fragment.

5. the method for claim 4, wherein said antibody fragment is an anti-TfR strand Fv fragment (TfRscFv).

6. the process of claim 1 wherein that described antibody or antibody fragment are anti--HER-2 antibody or antibody fragments.

7. the process of claim 1 wherein described antibody fragment with comprise the cysteine part at c-terminus before described cationic-liposome mixes.

8. the method for claim 1, further comprise with described cation immunoliposome with comprise K[K (H) KKK] peptide of 5-K (H) KKC (HoKC) (SEQ ID NO:1) peptide mixes.

9. the process of claim 1 wherein that described cationic-liposome comprises one or more lipids and one or more mixture neutral or auxiliary lipid.

10. the process of claim 1 wherein described antibody or antibody fragment and described cationic-liposome with about 1: 20 to about 1: 40 (w: mixed w).

11. the process of claim 1 wherein that described cationic-liposome comprises the mixture of two oleoyl front three ammonium phosphate and DOPE and/or cholesterol; Or the mixture of dimethyl two (octadecyl) ammonium bromide and DOPE and/or cholesterol.

12. the process of claim 1 wherein described cation immunoliposome and described preparation with about 0.1: 10 to about 0.1: 35 ratio (mg preparation: μ g liposome) mix.

13. the process of claim 1 wherein described cation immunoliposome and described preparation with about 1: 14 to about 1: 28 ratio (mg preparation: μ g liposome) mix.

14. the process of claim 1 wherein described cation immunoliposome and described preparation with about 1: 21 mol ratio (mg preparation: μ g liposome) mix.

15. the process of claim 1 wherein that described preparation is nuclear magnetic resonance (MRI) agent, computed tomography (CT) preparation or positron emission computerized tomography (PET) preparation.

16. the method for claim 15, wherein said MRI agent are gadolinium spray Portugal amine, ferrum oxide or iopamidol, and described CT preparation is a barium, iodine or saline, or described PET preparation is ¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG).

17. the cation immunoliposome complex that the method by claim 1 makes.

18. antibody or antibody fragment target cationic immunoliposome complex, it comprises cationic-liposome, antibody or antibody fragment and preparation, and wherein said antibody or antibody fragment are not chemically being puted together with described cationic-liposome.

19. the cation immunoliposome of claim 18, wherein said preparation is wrapped in the described cationic-liposome.

20. the cation immunoliposome complex of claim 18, wherein said preparation links to each other with the inboard or the outside monolayer of described cationic-liposome.

21. the cation immunoliposome complex of claim 18, wherein said antibody fragment are strand Fv fragments.

22. the cation immunoliposome complex of claim 18, wherein said antibody fragment are anti-TfR strand Fv (TfRscFv).

23. the cation immunoliposome complex of claim 18, wherein said antibody or antibody fragment are anti-HER-2 antibody or antibody fragment.

24. the cation immunoliposome complex of claim 18, wherein said cationic-liposome comprise one or more cationic-liposomes and one or more mixture neutral or auxiliary lipid.

25. the cation immunoliposome complex of claim 18, wherein said antibody or antibody fragment and described cationic-liposome were with about 1: 20 to about 1: 40 (w: ratio existence w).

26. the cation immunoliposome complex of claim 18, wherein said cationic-liposome comprises the mixture of two oleoyl front three ammonium phosphate and DOPE and/or cholesterol; Or the mixture of dimethyl two (octadecyl) ammonium bromide and DOPE and/or cholesterol.

27. the cation immunoliposome complex of claim 18, wherein said preparation and described cation immunoliposome with about 0.1: 10 to about 0.1: 35 ratio (mg preparation: μ g liposome) exist.

28. the cation immunoliposome complex of claim 18, wherein said preparation and described cation immunoliposome with about 1: 14 to about 1: 28 mol ratio (mg preparation: μ g liposome) exist.

29. the cation immunoliposome complex of claim 18, wherein said preparation and described cation immunoliposome are with about 1: 21 mol ratio (mg preparation: μ g liposome) exist.

30. the cation immunoliposome complex of claim 18, wherein said preparation are nuclear magnetic resonance (MRI) agent, computed tomography (CT) preparation or positron emission computerized tomography (PET) preparation.

31. the cation immunoliposome complex of claim 18, wherein said MRI agent are gadolinium spray Portugal amine, ferrum oxide or iopamidol, and described CT preparation is a barium, iodine or saline, or described PET preparation is ¹⁸F-2-deoxidation-2-fluoro-D-glucose (FDG).

32. the cation immunoliposome complex of claim 18 further comprises K[K (H) KKK that links to each other with described complex] 5-K (H) KKC (HoKC) (SEQ ID NO:1) peptide.

33. make patient organ or tissue imaging method, be included in the cation immunoliposome complex that carries out before the described imaging claim 18 and deliver medicine to the patient.

34. the method for claim 33, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, eye drops, intraperitoneal administration, administration in the tumor, administration or subcutaneous administration in the intranasal administration, brain.

35. make patient's cancerous tissue imaging method, be included in the cation immunoliposome complex that carries out before the described imaging claim 18 and deliver medicine to the patient.

36. make patient's cancerometastasis tumor imaging method, be included in the cation immunoliposome complex that carries out before the described imaging claim 18 and deliver medicine to the patient.

37. the method for claim 35, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, eye drops, intraperitoneal administration, administration in the tumor, administration or subcutaneous administration in the intranasal administration, brain.

38. the method for claim 36, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, eye drops, intraperitoneal administration, administration in the tumor, administration or subcutaneous administration in the intranasal administration, brain.

39. suffer from the patient's of cancer the imaging and the Therapeutic Method of tumor tissues, comprise that cation immunoliposome complex with claim 18 delivers medicine to the patient and comes the imaging tumor tissues and anticarcinogen is delivered medicine to the patient to treat tumor tissues.

40. the method for claim 39, wherein said administration comprises intravenous administration, intramuscular administration, intradermal administration, eye drops, intraperitoneal administration, administration in the tumor, administration or subcutaneous administration in the intranasal administration, brain.

41. the method for claim 39, wherein said anticarcinogen is a chemotherapeutics, gene or micromolecule.

42. the method for claim 41, wherein said chemotherapeutics is selected from amycin, mitoxantrone and gemcitabine.

43. the method for claim 41, wherein said anticarcinogen links to each other with described cation immunoliposome.

44. the method for claim 36, wherein said anticarcinogen are antisense oligonucleotide or siRNA.

45. the method for claim 44, wherein said antisense oligonucleotide or described siRNA link to each other with described cation immunoliposome.

46. the method for claim 39 wherein transmitted described anticarcinogen before or after cation immunoliposome complex.

47. the method for claim 46 wherein transmitted described anticarcinogen at least 12 hours before or after cation immunoliposome complex.

48. the method for claim 39 further comprises giving the patient radiotherapy.

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