KR101148979B1 - Biocompatible Cancer Cell Specific Delivery System - Google Patents

Abstract

The present invention relates to a cancer cell targeting virus whose surface is modified by a conjugate of a polymer-cancer cell targeting molecule exhibiting water-soluble natural polymer-anti-biofouling properties, and a pharmaceutical composition for treating cancer. The pharmaceutical compositions of the present invention comprising surface modified viruses significantly improve specific targeting to cancer cells, greatly increase cell permeation efficiency, and greatly reduce the immune response induced by viral vectors, thereby resulting in gene therapy for cancer. It is possible to achieve improvement, a breakthrough for. Through the properties of the surface modified virus described above, the surface modified virus of the present invention can be administered intravenously, which is a kind of systemic administration, which overcomes the disadvantages of local administration of conventional gene therapy agents. The surface modified virus of the present invention also enables repeated administration of gene therapeutics.

Adenovirus, Virus, Surface, Modified, Soluble natural polymer, Anti-biofouling, Cancer cell targeting molecule

Description

Biocompatible Cancer Cell Specific Delivery System

The present invention relates to biocompatible cancer cell specific carriers.

Adenovirus (ADV) has received a great deal of attention in the field of gene therapy because of its relatively large cloning capacity, ease of gene manipulation and proliferation and its ability to penetrate many tissues, including cloned and non-replicating cells. Is ^{[1, 2]} . Gene therapy is a powerful way to treat a variety of diseases. However, the absence of efficient vectors is an important problem in the development of the field of human gene therapy due to several problems such as instability in body fluids, nonspecificity to target cells, destruction by enzymes and low transformation efficiency ^[3] . However, viral vectors are used in clinical trials because of their high transformation efficiency compared to non-viral vectors.

On the other hand, chitosan, a non-viral polymer vector, is known as an excellent substance as a gene carrier. Chitosan is a natural cationic polysaccharide that has a variety of biomedical applications and properties, and is biocompatible and biodegradable; Binding ability to cells; Promoting wound healing; Hemostatic, antibacterial, antifungal and anticancer properties ^{[4, 5]} . Thus, chitosan derivatives are considered to be safe and efficient cationic carriers for gene delivery ^[6] .

Throughout this specification many patent documents and articles are referenced and their citations are indicated. The disclosures of cited patent documents and articles are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have attempted to develop a drug delivery system (DDS) capable of solving the clinical applicability of viral vectors as gene therapy, in particular, the challenge of inducing immune responses and nonspecific targeting by viral vectors and intravenously administering viral vectors. Efforts have shown that when the conjugate of the virus surface is coated with (i) a water-soluble natural polymer- (ii) a polymer exhibiting anti-biofouling properties- (iii) a cancer cell targeting molecule, the specific targeting to the cancer cells is greatly increased. The present invention has been completed by confirming that improved and cell transduction efficiency can be greatly increased, and that these properties enable intravenous administration of gene therapeutics.

It is therefore an object of the present invention to provide a biocompatible cancer cell specific carrier vehicle.

Another object of the present invention to provide a pharmaceutical composition for treating cancer.

It is another object of the present invention to provide a pharmaceutical composition for cancer imaging.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention provides a biocompatible cancer cell specific carrier vehicle of the virus as a gene carrier:

(a) a virus as a gene carrier; And

(b) a conjugate of (i) water soluble natural polymer- (ii) polymer exhibiting anti-biofouling properties coated on the surface of the virus- (iii) cancer cell targeting molecule.

The present inventors have attempted to develop a drug delivery system (DDS) capable of solving the clinical applicability of viral vectors as gene therapy, in particular, the challenge of inducing immune responses and nonspecific targeting by viral vectors and intravenously administering viral vectors. Efforts have shown that when the conjugate of the virus surface is coated with (i) a water-soluble natural polymer- (ii) a polymer exhibiting anti-biofouling properties- (iii) a cancer cell targeting molecule, the specific targeting to the cancer cells is greatly increased. It has been confirmed that the cell transduction efficiency can be improved and the cell transduction efficiency is greatly increased, and this property enables the intravenous administration of gene therapy products.

The surface of the virus of the invention is modified with conjugates of (i) water soluble natural polymers- (ii) polymers exhibiting anti-biofouling properties- (iii) cancer cell targeting molecules.

In the present invention, the conjugate for modifying the surface of the virus is composed of a water-soluble natural polymer, a polymer exhibiting anti-biofouling properties, and a cancer cell targeting molecule. In the surface modification conjugates of the present invention, water soluble natural polymers, anti-biofouling polymers and cancer cell targeting molecules can be bound in a variety of ways. For example, both an anti-biofouling polymer and a cancer cell targeting molecule can be combined to form a conjugate to a water-soluble natural polymer. In addition, the anti-biofouling polymer may be bound to the water-soluble natural polymer followed by the cancer cell targeting molecule to the anti-biofouling polymer.

Most preferably, the conjugate for surface modification is an anti-biofouing polymer bound to a water-soluble natural polymer followed by a cancer cell targeting molecule bound to the anti-biofouling polymer. The combination of the three materials can be achieved through the functional groups inherent in these materials, or can be combined by additionally introducing functional groups.

The water soluble natural polymers in the conjugate include any known in the art, preferably chitosan or derivatives thereof, dextran or derivatives thereof (eg carboxymethyl dextran), cellulose or derivatives thereof (eg carboxymethyl cellulose ), Heparin or derivatives thereof, hyaluronic acid or derivatives thereof, or alginate or derivatives thereof, more preferably chitosan, and most preferably low molecular weight chitosan (LMC).

As used herein, the term "low molecular weight chitosan" means an average molecular weight of 200-100,000, preferably an average molecular weight of 500-50,000, more preferably an average molecular weight of 1,000-50,000, even more preferably an average molecular weight of 1,000-10,000, most preferably Mean chitosan with an average molecular weight of 2,000-7,000.

According to a preferred embodiment of the present invention, the water-soluble natural polymer is alternatingly bonded by a crosslinking agent. This alternation allows the conjugate on the virus surface to be more tightly coated. In other words, the alternating bonds enable reticulation of surface modifiers comprising water soluble natural polymers. The crosslinking agents used in the present invention include any known in the art, preferably polyphosphate salts, most preferably sodium tripolyphosphate (TPP).

Water-soluble natural polymers, such as low molecular weight chitosan, are very useful for biocompatible modification of the viral surface to be transported and biodegradable in vivo .

Polymers exhibiting anti-biofouling properties used in the conjugates include anything known in the art, and preferably are PEG (polyethylene glycol), polyalkylene oxide, polyphenylene oxide, PEG and polyalkylene Copolymers of oxides, poly (methoxyethyl methacrylate), poly (methacryloyl phosphatidylcholine), perfluorinated polyethers or dextran, more preferably PEG.

The PEG used in the present invention is not particularly limited in molecular weight, and any PEG may be used. Preferably, PEG with a molecular weight of 300-30,000, more preferably of 500-10,000 of molecular weight and most preferably of 1,000-5,000 of molecular weight is used.

The anti-biofouling polymer used in the present invention prevents the virus of the present invention from being removed by the biofouling phenomenon when administered to the human body and acts to hide the virus from the immune response.

Modification by PEG, on the other hand, is a well-established technique for reducing protein-protein interactions between therapeutic agents, proteins and cells in vivo . In addition, PEGylation may increase the efficiency of infection into target cells because it results in prolonged circulation time in the bloodstream. PEGylation hides the virus from the immune response and can be used as a spacer (or linker) that connects cancer cell targeting molecules.

The cancer cell targeting molecule included in the conjugate allows the virus of the present invention to specifically infect cancer cells.

According to a preferred embodiment of the invention, the cancer cell targeting molecule is a ligand for a receptor of cancer cells or an antibody against a cancer cell surface antigen. Cancer cells are often characterized by specific antigens or overexpressing receptors on their cell surface. These receptors provide a route for receiving nutrients and signals from the surrounding environment. For example, the receptors for folic acid (FA), vitamin B ₁₂ and transferrin are overexpressed on the surface of cancer cells. By using a ligand that binds to the cancer cell overexpression receptor or an antibody against a cancer cell specific surface antigen, the drug can be specifically delivered to the cancer cell.

More preferably, the cancer cell targeting molecule is FA (folic acid), transferrin, vitamin B ₁₂ , methotrexate, vascular endothelial growth factor (VEGF), anti-vascular endothelial growth factor receptor (VEGFR) antibody, anti-HER2 / neu antibody , Anti-CEA (carcinoembryonic antigen) antibody, anti-PSA (Prostate specific antigen) antibody, anti-PSMA (prostate specific membrane antigen) antibody or anti-AFP (alpha-fetoprotein) antibody.

Most preferably, the cancer cell targeting molecule used in the present invention is FA (folic acid) (see SD Weitmann et al., Cancer Res . 52: 3396-3401 (1992); JF Ross et al., Cancer 73: 2432-2443 (1994); J. Holm et al., Biochem . J. 280: 267-272 (1991).

Receptors for FA are useful targets in cancer-specific gene delivery. FA is a high affinity ligand for the FA receptor (FR). FR is a 38 kDa glycosylphosphatidylinositol-anchor protein that binds to FA with very high affinity ^{[63, 64]} . Following binding, rapid endocytosis occurs and FA is transported into cells ^{[65, 66]} . Interestingly, when other molecules are covalently bound to the γ-carboxyl site of FA, the binding capacity of FA to FR is not changed and endocytosis occurs ^[67-69] . Among the isoforms of FRs (FR-α, FR-β and FR-γ), FR-α isoforms are mainly present in malignant tumors and various cancer cell lines ^{[75, 76]} . Aspects of overexpressed FR in human cancers are summarized in Table 1 below ^{[67, 80]} . Thus, FA conjugation is a very useful strategy for tumor-selective gene delivery ^{[65, 81]} .

cancer FR-positive ratio Ovarian Cancer 90% Brain cancer 75% Endometrial cancer 66% Kidney cancer 43% Head and Neck Cancer 38% Lung cancer 37% Breast cancer 32% Mesothelioma 73% Colon cancer 18%

The surface modification adopted in the present invention can be applied to various viruses, and preferably to viruses used as gene carriers in gene therapy. Preferably, the virus whose surface is modified is adenovirus (Lockett LJ, et al., Clin. Cancer Res . 3: 2075-2080 (1997)), Adeno-associated viruses: AAV, Lashford LS., Et al., Gene Therapy Technologies , Applications and Regulations Ed. A. Meager, 1999), retroviral (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies , Applications and Regulations Ed. A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin . Invest . 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc . Natl. Acad. Sci USA 92: 1411-1415 (1995)) or basinian virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)), more preferably, adenovirus, adeno-associated virus or retrovirus, most preferably adenovirus.

The virus of the present invention is basically prepared for treating cancer, and if necessary, the genome sequence of the virus is manipulated to insert a foreign gene suitable for the treatment of cancer. In this case, the foreign gene is preferably present in a suitable expression construct. In the expression construct, the foreign gene is preferably operably linked to the promoter. As used herein, the term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, thereby The regulatory sequence will control the transcription and / or translation of said other nucleic acid sequence. In the present invention, the promoter coupled to the foreign gene is preferably capable of controlling transcription of the foreign gene by operating in an animal cell, more preferably a mammalian cell, and a promoter and mammalian cell derived from a mammalian virus. Promoters derived from the genome of, for example, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk of HSV Promoter, RSV promoter, EF1 alpha promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene and human Including, but not limited to, promoters of the GM-CSF gene. Most preferably, it is a CMV promoter.

Preferably, the expression constructs used in the present invention comprise a polyaninylation sequence (eg, a glial growth hormone terminator and a SV40 derived poly adenylation sequence).

According to a preferred embodiment of the present invention, the foreign gene sequence used in the present invention has a structure of “promoter-foreign gene sequence-poly adenylation sequence”.

In the present invention, the foreign gene sequence to be transported into the cell may be any nucleotide sequence, for example, a cancer therapeutic gene that induces the death of cancer cells and ultimately degrades the tumor, and includes a tumor suppressor gene and an immunomodulatory gene (eg, cytokines). Genes, chemokine genes and costimulatory factors (adjuvant molecules required for T cell activity such as B7.1 and B7.2)], antigenic genes, suicide genes, cytotoxic genes, cytostatic genes, pro-cells Death genes and anti-angiogenic genes are included, but are not limited to these.

Suicide genes are nucleic acid sequences that express substances that cause cells to be killed by external factors or cause toxic conditions to cells. Well known such suicide genes are the thymidine kinase (TK) genes (US Pat. Nos. 5,631,236 and 5,601,818). Cells expressing the TK gene product are susceptible to selective killing by administration of gancyclovir. Tumor suppressor genes refer to genes encoding polypeptides that inhibit tumor formation. Tumor suppressor genes are naturally occurring genes in mammals, and it is believed that deletion or inactivation of these genes is an essential prerequisite for tumor development. Examples of tumor suppressor genes include APC, DPC4, NF-1, NF-2, MTS1, WT1, BRCA1, BRCA2, VHL, p53, Rb, MMAC-1, MMSC-2, retinoblastoma genes (Lee et al. Nature , 329: 642 (1987)), adenomatous polyposis coli protein (US Pat. No. 5,783,666), a throat disease suppressor gene located on chromosome 3p21.3 (Cheng et al. Proc . Nat . Acad . Sci . , 95: 3042-3047 (1998)), members of the INK4 family of tumor suppressor genes, including the missing colon tumor (DCC) gene, MTS1, CDK4, VHL, p110Rb, p16 and p21, and therapeutically effective fragments thereof (Eg, p56Rb, p94Rb, etc.). Those skilled in the art will appreciate that all other known anti-tumor genes besides the genes exemplified above may be used in the present invention.

As used herein, the term “antigenic gene” refers to a nucleotide sequence that is expressed in a target cell to produce cell surface antigenic proteins that can be recognized by the immune system. Examples of such antigenic genes include carcinoembryonic antigen (CEA) and prostate specific antigen (PSA), α-feto protein (AFP), p53 (WO 94/02167). To facilitate recognition of the immune system, the antigenic gene can be linked to an MHC type I antigen.

As used herein, the term "cytotoxic gene" refers to a nucleotide sequence that is expressed in a cell and exhibits a toxic effect. Examples of such cytotoxic genes include nucleotide sequences encoding Pseudomonas exotoxin, lysine toxin, diphtheria toxin, and the like.

As used herein, the term "cytostatic gene" refers to a nucleotide sequence that is expressed intracellularly and stops the cell cycle during the cell cycle. Examples of such cell proliferation inhibitory genes include p21, retinoblastoma gene, E2F-Rb fusion protein gene, genes encoding cyclin-dependent kinase inhibitors (eg, p16, p15, p18 and p19), growth stop specific homeo Growth arrest specific homeobox (GAX) genes (WO 97/16459 and WO 96/30385), and the like.

In addition, many therapeutic genes that can be usefully used to treat various diseases can also be carried by the virus of the invention. For example, cytokines (eg interferon-alpha, -beta, -delta and -gamma), interleukins (eg IL-1, IL-2, IL-4, IL-6, IL-7, IL-10 , Genes encoding IL-12, IL-19 and IL-20) and colony stimulating factors (eg, GM-CSF and G-CSF), chemokine groups (monocyte chemotactic protein 1 (MCP-1), monocyte chemotaxis) Protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 1β (MIP-1β), Macrophage inflammatory protein 1γ (MIP-1γ), macrophage inflammatory protein 3α (MIP-3α), macrophage inflammatory protein 3β (MIP-3β), chemokine (ELC), macrophage inflammatory protein 4 (MIP-4), macrophage inflammatory protein 5 ( MIP-5), LD78β, RANTES, SIS-epsilon (p500), thymus activation-regulated chemokine (TARC), eotaxin, I-309, human protein HCC-1 / NCC-2, human protein HCC-3, mouse Protein C10, etc.). Also included are genes expressing tissue plasminogen activator (tPA) or urokinase and LAL generating genes that provide sustained thrombotic effects to prevent cholesterol hyperemia. In addition, many polynucleotides are known for treating viral, malignant and inflammatory diseases and conditions such as cystic fibrosis, adenosine deaminase deficiency, and AIDS.

As used herein, the term “pro-apoptotic gene” refers to a nucleotide sequence that is expressed and induces programmed cell death. Examples of such pro-apoptotic genes include p53, adenovirus E3-11.6K (derived from Ad2 and Ad5) or adenovirus E3-10.5K (derived from Ad), adenovirus E4 gene, Fas ligand, TNF-α, TRAIL genes encoding the p53 pathway gene and caspase.

As used herein, the term "anti-angiogenic gene" refers to a nucleotide sequence that is expressed and releases an anti-angiogenic factor extracellularly. Anti-angiogenic factors include angiostatin, inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2 (PNAS, 1998, 95,8795-800), endostatin and the like.

The nucleotide sequence of the foreign gene described above can be obtained from a DNA sequence database such as GenBank or EMBL.

According to the most preferred embodiment of the present invention, the gene carrier used in the present invention is an adenovirus.

Adenoviruses are widely used as gene transfer vectors because of their medium genome size, ease of manipulation, high titers, wide range of target cells and excellent infectivity. Both ends of the genome contain 100-200 bp of Inverted Terminal Repeat (ITR), which is an essential cis element for DNA replication and packaging. Because only a small part of the adenovirus genome is known to be needed in cis (Tooza, J. Molecular biology of DNA Tumor viruses , 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1981)), anenoviruses are capable of carrying large quantities of foreign DNA molecules, especially when using certain cell lines such as 293. In this aspect, the adenoviruses used in the present invention, in addition to the foreign therapeutic gene, the sequences of other adenoviruses include at least an ITR sequence.

The genome El region (E1A and E1B) encodes proteins that regulate transcription and transcription of host cell genes. The E2 regions (E2A and E2B) encode proteins that are involved in viral DNA replication.

Among the adenovirus vectors currently developed, non-replicating adenoviruses lacking the E1 region are widely used. On the other hand, the E3 region is removed from conventional adenovirus vectors to provide a site for insertion of foreign genes (Thimmappaya, B. et al., Cell , 31: 543-551 (1982); and Riordan, JR et al., Science , 245: 1066-1073 (1989).

Therefore, the foreign therapeutic gene is preferably inserted into the deleted E1 region (E1A region and / or E1B region) and / or E3 region.

According to a preferred embodiment of the invention, the recombinant adenovirus of the present invention is a nucleotide sequence in which the E1B gene is deleted and which encodes an Rb binding site in the E1A gene sequence (ie, CR1, CR2 or a nucleotide sequence encoding CR1 and CR2). It is an adenovirus in which mutation occurs and the binding ability with Rb is lost. More preferably, the 45th Glu residue of the ElA protein is substituted with Gly, the 121-127 amino acid sequence is entirely substituted with Gly, and the 124th Cys residue is substituted with Gly. Details of recombinant adenoviruses that have lost such RB binding capacity are disclosed in our patent application 2004-0032638 (KCCM-10569).

Meanwhile, the insertion sequences can also be inserted into the deleted E4 region. As used herein, the term "deletion" as used in connection with a viral genome sequence has the meaning including not only a complete deletion of the sequence, but also a partial deletion.

In addition, because adenovirus can pack up to about 105% of the wild-type genome, about 2 kb can be additionally packaged (Ghosh-Choudhury et al., EMBO J. , 6: 1733-1739 (1987)). Thus, the above-described foreign sequence inserted into the adenovirus may additionally bind to the genome of the adenovirus.

As used herein, the term “adenovirus” is used in the same way as an “adenovirus vector” and means a virus belonging to the genus “Adenoviridiea”. The term 'adenoviridiea' refers collectively to, but is not limited to, animal adenoviruses of the genus Mastadenovirus (including, but not limited to, human, bovine, sheep, horse, dog, pig, rat and monkey adenovirus subgenus). . In particular, human adenoviruses comprise the AF subgenra and their respective serotypes, and AF subgenus of human adenoviruses type 1, type 2, type 3, type 4, type 4a, Type 5, Type 6, Type 7, Type 8, Type 9, Type 10, Type 11 (Ad11A and Ad11P), Type 12, Type 13, Type 14, Type 15, Type 16 Type 17, Type 18, Type 19, Type 19a, Type 20, Type 21, Type 22, Type 23, Type 24, Type 25, Type 26, Type 27, Type 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38 Types 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 and 91, including but not limited to It doesn't happen. The adenovirus in a preferred embodiment of the present invention is derived from human adenovirus second or fifth serotypes, and most preferably from the fifth serotype.

Foreign therapeutic genes carried by adenoviruses replicate in the same way as episomes, and have very low genetic toxicity to host cells. Therefore, gene therapy using the adenovirus vector of the present invention is considered to be very safe.

Meanwhile, the genome of the virus of the present invention may further include a relaxin gene or a decorin gene in order to maximize gene transfer efficiency. Improvement of gene transfer efficiency by the relaxin gene or decorin gene is disclosed in detail in our patent applications 2005-0026808 and 2006-0094630.

The surface-modified virus of the present invention significantly improves the specific targeting of cancer cells, greatly increases the cell permeation efficiency, and greatly reduces the immune response induced by viral vectors, thereby dramatically improving gene therapy for cancer. Bring.

According to a preferred embodiment of the invention, the delivery vehicle of the invention is a delivery vehicle for intravenous administration. Although most gene therapy viruses are administered locally, the carrier vehicle of the present invention can be administered intravenously by surface modification and can exhibit sufficient therapeutic efficacy by intravenous administration.

The biocompatible cancer cell specific carrier vehicles of the present invention can be prepared with nanoparticles according to various methods known in the art, such as by electrospinning. In this case, the carrier vehicle of the invention preferably has a size of 10-500 nm, more preferably of 50-300 nm and most preferably of 100-200 nm.

The apparatus for carrying out electrospinning is divided into three parts (see Fig. 1): a high voltage power supply, a spinneret (metallic needle) and a collector (grounding conductor). Although an AC power supply may be used, it is preferable to use a DC power supply in the present invention. The spinneret is connected to a syringe, which contains a solution of surface modification material and a virus solution. A syringe pump is used to allow the solution to pass through the spinneret, but at a constant rate. When high pressure is applied, the drops of the solution in the spinneret's nozzles are highly electromagnetized and the induced charge is evenly distributed throughout the surface.

According to a preferred embodiment of the present invention, the voltage applied during electrospinning is 10-20 kV, more preferably 13-17 kV. The flow rate through the spinneret is preferably 1-10 mL / hr, more preferably 3-7 mL / hr. The gauge of the metal needle used as the spinneret is preferably 20-50 gauge, more preferably 20-40 gauge.

When the carrier vehicle of the present invention is prepared by the electrospinning method, it may include the following steps: (a) (i) water-soluble natural polymers- (ii) polymers exhibiting anti-biofouling properties- (iii) cancer cells Placing a mixed solution of a solution of the conjugate of the targeting molecule and a virus solution into the syringe; (b) passing the mixed solution under a voltage of 10-20 kV at a flow rate of 3-7 mL / hr through a spinneret consisting of 20-50 gauge metal needles; (c) collecting the passed drop-form solution in a collector; And (d) subjecting the collected solution to an alternating reaction in the presence of an alternating binder.

According to another aspect of the invention, the invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the biocompatible cancer cell specific carrier vehicle described above; And (b) provides a pharmaceutical composition for treating cancer comprising a pharmaceutically acceptable carrier.

Since the pharmaceutical composition of the present invention uses the above-described surface modified virus of the present invention as an active ingredient, the contents common to both are omitted in order to avoid excessive complexity of the present specification.

In order to induce an effective anti-tumor effect with a virus, the virus must infiltrate tumor cells in vivo specifically and with high efficiency. The surface modified virus developed in the present invention increases the specific binding of the virus to cancer cells, penetrates the cells with high efficiency, and greatly increases the efficacy of cancer treatment by minimizing the immune response by the viral vector. .

The virus included in the pharmaceutical composition for treating cancer of the present invention preferably includes an outpatient therapeutic gene.

As used herein, the term “treatment” means (i) prevention of tumor cell formation; (Ii) inhibition of a disease or condition associated with the tumor following removal of the tumor cells; And (iii) alleviation of a disease or disorder associated with a tumor following removal of tumor cells. Thus, the term "pharmaceutically effective amount" herein means an amount sufficient to achieve the above pharmacological effect.

Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition of the present invention is preferably parenteral, and may be administered using, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration. Preferably, the pharmaceutical composition of the present invention is administered intravenously. The surface modified virus of the present invention enables specific targeting to cancer cells and greatly reduces immunogenicity, thereby allowing intravenous administration, which is a type of systemic administration. Such systemic administration allows to overcome the disadvantages of topical administration of conventional gene therapy agents.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. In general, the pharmaceutical compositions of the present invention comprise 1 × 10 ⁵ -1 × 10 ¹⁵ pfu / ml of virus, and typically 1 × 10 ¹⁰ pfu is injected for two weeks, once every two days.

The pharmaceutical compositions of the present invention are prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The pharmaceutical composition of the present invention may be used as a single therapy, but may also be used in combination with other conventional chemotherapy or radiation therapy, and when the combination therapy is performed, cancer treatment may be more effectively performed. Chemotherapeutic agents that can be used with the compositions of the present invention are cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, phospho Ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin, daunorubicin, doxorubicin , Bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluoro 5-urauracil, vincristin, vinblastin, methotrexate and the like. Radiation therapy that can be used with the composition of the present invention is X-ray irradiation, γ-ray irradiation, and the like.

According to another aspect of the present invention, the present invention provides a composition for cancer imaging, comprising: (a) a pharmaceutically effective amount of the biocompatible cancer cell specific carrier vehicle described above; And (b) a pharmaceutically acceptable carrier.

Since the composition for cancer imaging of the present invention uses the above-described surface modified virus of the present invention as an active ingredient, the contents common between the two are omitted in order to avoid excessive complexity of the present specification.

Because the carrier vehicle of the present invention is selectively targeted to cancer cells, it can be used to image cancer tissues and cells.

According to a preferred embodiment of the present invention, the virus included in the composition for cancer imaging of the present invention comprises a gene encoding a fluorescent protein (eg, green fluorescent protein).

According to a preferred embodiment of the present invention, the virus included in the composition for cancer imaging of the present invention is further combined with an imaging agent (imaging agent). The imaging agent may be bound to a water soluble natural polymer, an anti-biofouling polymer or a cancer cell targeting molecule. The imaging agent is a radioisotope (e.g., ¹⁰ C, ¹¹ C, ¹³ O, ¹⁴ O, ¹⁵ O, ¹² N, ¹³ N, ¹⁵ F, ¹⁷ F, ¹⁸ F, ³² Cl, ³³ Cl, ³⁴ Cl, ⁴³ Sc, ⁴⁴ Sc, ⁴⁵ Ti, ⁶⁶ Ge, ⁶⁷ Ge, ⁶⁸ Ga, ⁶⁹ Ge, ⁶⁹ As, ⁷⁰ As, ⁷⁰ Se, ⁷¹ Se, ⁷¹ As, ⁷² As ⁷³ Se, ⁷⁴ Kr, ⁷⁴ Br, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁷⁷ Kr, ⁷⁸ Br, ⁷⁸ Rb, ⁷⁹ Rb, ⁷⁹ Kr, ⁸¹ Rb, ⁸² Rb, ⁸⁴ Rb, ⁸⁴ Zr, ⁸⁵ Y, ⁸⁶ Y, ⁸⁷ Y, ⁸⁷ Zr, ⁸⁸ Y, ⁸⁹ Zr, ⁹² Tc, ⁹³ Tc, ⁹⁴ Tc, ⁹⁵ Tc, ⁹⁵ Ru, ⁹⁵ Rh, ⁹⁶ Rh, ⁹⁷ Rh, ⁹⁸ Rh, ⁹⁹ Rh, ¹⁰⁰ Rh, ¹⁰⁸ In, ¹⁰⁹ In, ¹¹⁰ In, ¹¹⁵ Sb, ¹¹⁶ Sb, ¹¹⁷ Sb , ¹¹⁵ Te, ¹¹⁶ Te, ¹¹⁷ Te, ¹¹⁷ I, ¹¹⁸ I, ¹¹⁸ Xe, ¹¹⁹ Xe, ¹¹⁹ I, ¹¹⁹ Te, ¹²⁰ I, ¹²⁰ Xe, ¹²¹ Xe, ¹²¹ I, ¹²² I, ¹²³ Xe, ¹²⁴ I, ¹²⁶ I, ¹²⁸ I, ¹³¹ I, ¹²⁹ La, ¹³⁰ La, ¹³¹ La, ¹³² La, ¹³³ La, ¹³⁵ La, ¹³⁶ La, ¹⁴⁰ Sm, ¹⁴¹ Sm, ¹⁴² Sm, ¹⁴⁴ Gd, ¹⁴⁵ Gd, ¹⁴⁵ Eu, ¹⁴⁶ Gd, ¹⁴⁶ Eu, ¹⁴⁷ Eu, ¹⁴⁷ Gd, ¹⁴⁸ Eu, ¹⁵⁰ Eu, ¹⁹³ Tl, ¹⁹⁴ Tl, ¹⁹⁴ Au, ¹⁹⁵ Tl, ¹⁹⁶ Tl, ¹⁹⁷ Tl, ¹⁹⁸ Tl, ²⁰⁰ Tl, ²⁰⁰ Bi, ²⁰² Bi, ²⁰³ Bi, ²⁰⁵ Bi , ²⁰⁶ Bi, or derivatives thereof, fluorescent materials (eg, fluorosane, rhodamine, lucifer yellow) , B-phytoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilben-2,2'-disulfonic acid, 7-die Ethylamino-3- (4'-isothiocyatophenyl) -4-methylcoumarin, succinimidyl-pyrenebutyrate, 4-acetamido-4'-isothiocyanatostilbene-2,2'- Disulfonic acid derivatives, LC ™ -Red 640, LC ™ -Red 705, Cy5, Cy5.5, lysamine, isothiocyanate, erythrosine isothiocyanate, diethylenetriamine pentaacetate, 1-di Methylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidiyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, 9-isothio Cyanatoacridin, acridine orange, N- (p- (2-benzoxazoylyl) phenyl) melimide, benzoxadiazole, stilbene, pyrene, derivatives thereof), group II / VI Semiconductor Quantum Dots, Group III / V Semiconductor Quantum Dots, Group IV Comprises a conductor quantum dots, gold nanoparticles, silver nanoparticles, and portrait midnight nanoparticles (e.g., magnetic iron oxides (e.g., _{magnetite, Fe 3 O 4), γ} -Fe 2 O 3, manganese ferrite, cobalt ferrite and nickel ferrite) do.

According to another variant of the invention, the composition of the invention can be prepared to be capable of simultaneously treating and imaging cancer. For example, incorporating a foreign therapeutic gene into a virus and incorporating the above-described imaging agent on the surface of the virus allows simultaneous treatment and imaging of cancer.

The features and advantages of the present invention are summarized as follows:

(Iii) The surface modified virus of the present invention is surface modified by the conjugate of a polymer-cancer cell targeting molecule exhibiting water-soluble natural polymer-anti-biofouling properties.

(Ii) The cancer cell targeting molecule conjugated to the virus of the present invention helps to specifically target the viral vector to tumor cells, and polymers exhibiting anti-biofouling properties such as PEG can be used to administer the virus of the present invention to the human body. In this case, the biofouling effect is prevented from being removed and the virus is masked from the immune response, and the water-soluble natural polymer functions to greatly improve the biocompatibility of the carrier carrier of the present invention.

(Iii) In conclusion, the pharmaceutical compositions of the present invention comprising surface modified viruses have a markedly improved specific targeting to cancer cells, a significant increase in cell permeation efficiency, and a significant reduction in immune responses induced by viral vectors. It is possible to achieve a breakthrough in gene therapy for cancer.

(Iii) In addition, the virus of the present invention can exert pharmacological efficacy at a lower dosage, and its efficacy can be maintained for a longer period of time.

(v) Through the characteristics of the surface modified virus described above, the surface modified virus of the present invention can be administered intravenously, which is a kind of systemic administration, which overcomes the disadvantages of local administration of conventional gene therapy agents.

(vi) The surface modified virus of the present invention also allows repeated administration of a gene therapy agent.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Materials and Experimental Methods

Experimental material

A water soluble low molecular weight chitosan (weight basis molecular weight, 3000-5000) was purchased from KITTOLIFE Co. (South Korea). Hetero-bifunctional poly (ethylene glycol) derivative (NH ₂ -PEG-COOH, Mw 3400) was purchased from Laysan Bio (Laysan Bio Inc. USA). Membrane was purchased from Membrane-mfpi (Cellu Sep T3, MWCO 3500, USA). DPBS (1 ×), tripolyphosphate (TPP), N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide (EDC), di-cyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHC) were purchased from Sigma Aldrich.

Cell line and cell culture

Human epithelial cancer cell lines were cultured in folate-deficient RPMI 1640 (Gibco BRL, cat.27016) from oral KB cells overexpressing folate receptors (Korea Cell Line Bank) to induce overexpression of folate receptors on KB cell membranes. Human glioma brain tumor U343 cells (ATCC) without folate receptors were cultured in DMEM (HyClone, Logan, UT, USA). Each cell medium contains 10% fetal bovine serum, penicillin and streptomycin 100 IU / ml. Cells were seeded in 24-well plates. After 24 hours, each cell was infected with ADV, ADV-cLMC, ADV-LMC-FA, ADV-LMC-PEG and ADV-LCPF. Cells were incubated at 37 ° C. for 48 hours, then GFP expression in infected cells was measured by fluorescence microscopy (Olympus BX51; Olympus Optical, Tokyo, Japan) and subjected to FACS analysis.

GFP ( Green fluorescent protein Expression ADV Manufacturing

The ADV used is E1-deficient Ad type 5 expressing GFP under the control of the cytomegalovirus (CMV) promoter at the E1 site of adenovirus. The manufacturing process is as follows: To develop a non-replicating adenovirus expressing GFP, pEGFP-N1 (Clontech) was cut to NotI, followed by treatment with T4 DNA polymerase to blunt the DNA ends, which were then treated with XhoI. The resulting 800 bp DNA fragment was ligated with the pCA14 vector (Microbix) treated with XhoI and EcoRV to construct a pCA14 / EGFP shuttle vector. The produced shuttle vector was cut again with XmnI and dl324BstBI DNA (Brca, Dr. Verca, Fribourgh University, Switzerland; Heider, H. et al., Biotechniques , 28 (2): 260-265, 268-270 (2000); Finally, adenovirus vectors containing GFP were prepared by homologous recombination in KFCC-11280, KFCC11288) and BJ5183 (ATCC). The produced adenovirus vector was digested with Hind III and PacI enzymes to verify DNA, and then transfected with PacI to HEK293A cell line (Microbix) to observe virus production. Thus prepared ADV expressing GFP was propagated in 293 cells (Microbix) and purified according to CsCl density gradient purification. Purified viion was dialyzed with 10 mM Tris and stored at -80 ° C. The number of virus particles was calculated by measuring the optical density at 260 nm (OD ₂₆₀ ), and one absorbance unit corresponds to 10 ¹² virus particles (VP) / ml.

ADV - cLMC  particle

One) LMC  And TPP  Preparation of the solution

Each powder was dissolved and stirred at room temperature in IX PBS (phosphate-buffered saline) solution to prepare a water-soluble, low molecular weight chitosan and TPP solution. The final concentration of chitosan was 1.0 wt% and the final concentration of TPP was 1.0 wt%.

2) by electrospinning ADV - cLMC Manufacturing

A 1 mL LMC mixed ADV solution was passed at a flow rate of 5 mL / hr through a nozzle with a 30-gauge blunt needle using an electrospinning (NNC-ESP 200, Nano NC, Seoul, Korea) syringe pump. The solution contains 60 μl ADV, ie 1 × 10 ¹² VP / ml. A 3 mL crosslinker, TPP, was connected at the bottom. The distance between the tip-to-collectors was kept at 5 cm and the voltage was 15 kV. After electrospinning, dialysis was performed and purified by ultrafiltration using Centricon 50-membrane tubes (Amicon, MW cutoff: 10,000) to remove LMC residue. Purified ADV-cLMC (crosslinked LMC) was stored at -20 ° C.

The electrospinning apparatus is schematically shown in FIG.

3) Measurement of average size and surface charge

The sizes of ADV, LMC and ADV-cLMC were measured using a DLS (Zeta-potential Analyzer ELS-Z, Photal, Otsuka, Japan). Each sample had a volume of 2 mL. Their surface charge was also measured (Zetasizer 3000HS). The charge of ADV was weakly negative. For the ζ-potential measurement, each of ADV, LMC, ADV-cLMC and ADV-LMC-FA was dissolved in deionized water (1.0 mg / ml) and measured at 25 ° C.

4) Chitosan Analysis

The degree of binding between chitosan and TPP was determined by measuring the amount of free amino groups of chitosan by fluorescamine analysis before and after complexation using TPP. 1 mL of nanoparticle solution was added to 14 mL of 100 mM boric acid-NaOH (pH 8) in a 96-well plate. Wells containing only boric acid buffer were used to determine background fluorescence. 5 mL of 0.01% Fluorskamin (Sigma-Aldrich) solution prepared in PBS was added to each well, mixed and reacted for 20 minutes at room temperature. Fluorescence was measured with an ELISA reader (Vmax, Molecular devices, Sunnyvale, CA, USA) with an excitation wavelength of 390 nm and emission wavelength of 475 nm.

LCPF Characteristic study of

One) PEG - FA Synthesis of

FA (folic acid, Sigma-Aldrich) was activated with di-cyclohexylcarbodiimide (DCC) and N- hydroxysuccinimide (NHS) for 12 hours in a DMSO at a molar ratio of 1: 2: 2 under a nitrogen atmosphere, followed by a syringe filter Size, 0.2). PEG derivatives (NH ₂ -PEG-COOH, MW 3400) were added to the solution in a molar ratio of 15: 1 (FA: PEG). Under nitrogen atmosphere, the reaction was carried out for 4 hours and then the reaction was precipitated with acetone and centrifuged to remove free FA residue. The supernatant FA-PEG conjugate was dialyzed with deionized water (SpectraPor6, MW cutoff: 1000) and lyophilized.

2) LMC - PEG - FA ( LCPF ) Synthesis

To bind PEG-FA to LMC, LMC was impregnated with 1.0 wt% PEG-FA aqueous solution followed by addition of EDC and NHS at room temperature. The solution has a LMC / PEG-FA feed weight ratio of 1: 1 and a PEG-FA / EDC / NHS molar ratio of 2: 2: 1. LMC in PEG-FA solution was stirred slowly at room temperature overnight. After surface grafting as above, PEG-FA residue that did not react with LMC amino group was removed by dialysis with deionized water (SpectraPor6, MW cutoff: 1000) and the result was lyophilized.

3) FT - IR ( Fourier transform infra - red ) Spectroscopy

The chemical structure of the -CONH functional groups of LCPF was analyzed by FT-IR spectroscopy (TENSOR 27, Bruker Optik GmbH, Germany) in the range of 400-4000 cm ^-1 at room temperature. FT-IR spectra were obtained by the KBr method. For the measurement, the samples were embedded in KBr pellets (sample: KBr weight ratio = 1: 100). The blank background was removed from all spectra.

4) ^One H- NMR ( ^One H- Nuclear magnetic resonance ) Spectroscopy

To identify the functional groups, ¹ H-NMR spectra for LCPF particles were recorded on a 500 MHz NMR spectrometer (AVANCE 500, Bruker, Germany). For the measurement, LCPF samples were dissolved in a DCl / D ₂ O mixture (1 mL D ₂ O in 1 drop DCl solution).

ADV - LCPF  particle

1) by electrospinning ADV - LCPF Manufacturing

1 mL of PBS and 1 mg of synthesized LCPF were mixed in an Eppendorf-tube, followed by vortexing for 60 seconds. 60 μl of 1 × 10 ¹² VP / ml ADV was added and electrospinning. Electrospinning was carried out under the conditions described in FIG. 1, and then alternating was performed for 20 minutes. The result was dialyzed and purified by ultrafiltration using Centricon 50-membrane tube (Amicon, MW cutoff: 10,000). As LCPF virus camp passes through the needles, particles are formed by amide bonds of NH _{2 of} LMC and O of TPP. Chitosan coatings act to protect the surface of viral particles, while external PEG and folate are involved in cancer targeting.

2) TEM ( transmission electron microscopy Shape and size analysis

The surface morphology and internal structure of ADV, ADV-cLMC, ADV-LMC-PEG and ADV-LCPF were observed using a JEM 1011 transmission electron microscope (JEOL, Nikon, Japan) (at 80 kV). Before observation, ADV and ADV-cLMC were added to glutaraldehyde in a volume ratio of 2: 1 and negatively stained with phosphotungstic acid.

FACS In Vitro Cell Expression Analysis by

To quantitatively measure GFP expression levels in ADV, cancer cells were infected with ADV and electrospun ADV. KB and U343 cells were seeded in 24-well plates at a density of 1 × 10 ⁵ cells / well, and the cells were infected with ADV and the like. 48 hours after infection, cells were isolated with lysate (Sigma, St Louis, MO, USA) and washed three times with PBS. After washing, 500 μl of PBS was added, followed by fluorescence activated cell sorting (FACS) analysis using FACScan (Beckton-Dickinson, Sunnyvale, Calif., USA) equipped with cell quest software (Beckton-Dickinson).

Experiment result

One. ADV , LMC  And ADV - cLMC  Surface charge

Viral surface charges generally have a weak negative charge. LMC is a cationic polymer and has a positive charge. ADV is coated with LMC and some of the LMC amines are bound to oxygen ions in TPP. Thus, the charge of ADV-cLMC has a lower positive charge than the charge by LMC alone. Thus, the present inventors measured the ζ-potential. As can be seen in Figure 2, ADV was negative charge, and ADV-cLMC showed a positive charge.

2. LMC  coating ADV

One) LMC Morphological analysis of various concentrations of

As can be seen in (a) and (f) TEM images of FIG. 3, ADV showed a size of about 55 nm and a clear surface. ADV core particles were incorporated into the LMC layer, LMC and TPP interaction. The ADV-0.5 wt% cLMC was about 63.6 nm, the ADV-2.0 wt% cLMC was about 68.0 nm, and the layer thickness around ADV was 10 nm. The ADV-cLMC surface turned rough. As can be seen from the TEM image, the ADV-cLMC size increased after LMC coating.

2) various concentrations LMC Coated by ADV  Size change

As can be seen in Figure 4, the size pattern measured by the DLS is consistent with that measured in the TEM. It can be seen that the ADV-cLMC size can be controlled by the LMC concentration. As the LMC concentration increased from 0.0 to 1.0 wt%, the ADV-cLMC size also increased rapidly. However, at the LMC concentration of 1.0 wt% to 2.0 wt%, the ADV-cLMC size was hardly changed. Thus, 1.0 wt% of LMC was determined at a concentration suitable for coding ADV.

3) LMC  Surface tension and viscosity of the solution

Table 2 shows the measured surface tension and viscosity of the LMC.

LMC conc.
(wt%) Viscosity
(cp) Surface tension
(dyne / cm) 0.5 1.2 74.92 One 1.3 73.24 1.5 1.8 72.42 2 2.4 70.98

This shows the properties of the LMC solution. As the LMC concentration increased from 0.0 wt% to 2.0 wt%, the surface tension decreased to 74.9 to 70.6 dyne / cm and there was a significant change in viscosity. On the other hand, the viscosity was increased to 1.2 to 2.4 cp. Thus it can be seen that increasing the LMC concentration increases the charge density on the surface of the emission jet in the electrospinning; The increase in charge density carried by the jet is due to the stronger whipping instability of the jet. Thus surface tension and viscosity do not affect the critical voltage.

4) by chitosan analysis LMC Validity of NH ₂

In order to determine the optimal coating conditions, the degree of binding between LMC and TPP at different LMC concentrations should be evaluated. Fluorskamin assay (chitosan assay) was used to determine the free amino groups on the polymer. This analytical method is very useful because it does not interact with amino groups that are cross-linked with TPP. Thus, after cross-linking with TPP, free amino groups on the LMC can be quantified. Experimental results are described in FIG. 5. As expected, the free amino group of ADV-cLMC increased with increasing LMC concentration. According to Figure 5, it can be seen that the concentration of LMC of 1 wt% is sufficient for PEG bonds and TPP alternating bonds.

3. LCPF Characteristics study for

One) PEG - FA FT - IR  And ^One H- NMR spectroscopy

PEG, FA, and PEG-FA conjugates were identified by FT-IR (FIG. 6A). COC stretching at 1106 cm ¹ ; Anti-symmetric stretching, CH out-of-plane bending at 1344 cm ¹ and 966 cm ¹ indicates that PEG is chemically bound. The FT-IR spectrum modified with FA in FIG. 6A shows the characteristic IR absorption peak of FA at 1700 cm ¹ .

As can be seen in Figure 6b, peaks were observed for PEG (3.4 ppm), FA (6.5, 7.4 ppm). Peaks for the ethylene repeat units of PEG were observed at 3.48 ppm. The molecular structure of the conjugate was confirmed through the presence of all characteristic peaks. The experimental results above indicate that FA is bound to the PEG backbone.

2) LCPF FT - IR  And ^One H- NMR Spectroscopy

The IR spectrum of the LMC is shown in Figure 7a. Peaks at 1640, 1530 cm ⁻¹ show strong NH bending vibrations of the primary and secondary amides. The peak at 1700 cm ⁻¹ in the IR spectrum of FA corresponds to the strong COOH and the peak at 1640 cm ^{−1 corresponding} to the aromatics C═C and CC. In the IR spectrum of the LCPF copolymer, the new peak appearing at 1310 cm ⁻¹ indicates the presence of secondary amide bonds of LMC-PEG and the low peak intensity at 1700 cm ⁻¹ corresponds to PEG-FA. These results show that LMC-PEG-FA is bound to the LMC backbone. In FIG. 7B, the ¹ H NMR spectrum of 35 wt% DCl in D ₂ O shows the proton signal of OCH ₂ CH ₂ (PEG chain). In particular, the conjugates exhibit the absence of a signal of an amino group (δ = 7.3 ppm). In addition, the ¹ H NMR analysis can confirm that the hydroxyl group in the partial polymer bond.

4. Coated with various materials ADV Size

8 shows the DLS measured sizes of ADV coated with various materials, showing the expected sizes for ADV, ADV-cLMC, ADV-LMC-FA, ADV-LMC-PEG and ADV-LCPF, respectively. Overall, the DLS data was very similar to the TEM image. The ADV size was about 50 nm and the ADV-cLMC, ADV-LMC-FA, ADV-LMC-PEG and ADV-LCPF sizes were about 55, 61, 136 and 169 nm, respectively.

variety LMC  Coated with concentration ADV  Particles Intracellular  infection

One) ADV - cLMC Fluorescence microscopy analysis for

9 is a fluorescence microscope image of GFP expression in U343 cells. 1 wt% of TPP was used and various concentrations of LMC were used. As LMC concentration increased, GFP expression also increased. At the LMC concentration of 1.0 wt%, the degree of GFP expression peaked. Thus, it can be seen that a suitable LMC coating concentration is 1 wt%.

2) ADV - cLMC of FACS  analysis

To quantify the expression level of GFP in ADV, FACS analysis was performed. When the FACS data were graphed, the FACS results were nearly identical to the transformation efficiency results. As can be seen in Figure 10, the uncoated ADV showed the lowest expression level, the expression level continuously increased up to 1 wt% LMC concentration.

6. KB  And U343  In a cell GFP  Expression degree

1) coated with various materials ADV of GFP  Expression

Fluorescence microscopy and FACS analysis were used to determine whether FR-specific nanoparticles enter KB cells and U343 cells. KB cells and U343 cells are FR overexpressing cells and FR-deficient cells, respectively. FIG. 13A shows the results obtained by fluorescence microscopy observation of 30 MOI particles treated at 37 ° C. for 48 hours. FIG. 13B is a FACS result.

Folate conjugated nanoparticles inhaled into KB cells showed more green than U343 cells. The level of GFP expression in KB cells increased in the order of ADV, ADV-cLMC and ADV-LCPF. However, the expression level of ADV-LCPF in U343 cells was almost the same as that of ADV-LMC-PEG. On the other hand, in both KB cells and U343 cells, the expression level of GFP of ADV-LMC-PEG was lower than that of ADV, because PEG chain acts to coat knob protein for ADV cell infection. Comparing the results of KB cells and U343 cells, intracellular uptake of folate-conjugated ADV-LCPF nanoparticles is achieved by binding to FR overexpressed in KB cells, and thus cells of genes carried by ADV. It can be seen that the endogenous expression can be specifically made of cancer cells overexpressed FR.

2) according to various folate concentrations ADV - LCPF  Particle GFP  Expression analysis

12 and 13 show the degree of GFP expression in KB cells and U343 cells. Cells were treated with 50 MOI at 37 ° C. and subjected to fluorescence microscopy and FACS analysis 48 hours later. As FA concentration of ADV-LCPF increased from 0.0% to 20.0%, GFP expression increased concentration dependent because KB cells were FR overexpressing cells. As a result, it can be seen that the folate of ADV-LCPF targets FR. In contrast, for U343 cells, an increase in FA concentration rather reduced GFP expression. In both KB cells and U343 cells, at least 30% FA resulted in low GFP expression. This is because the excessive concentration of FA in ADV-LCPF causes steric hindrance effect.

Methods for delivering gene carriers to cancer cells include direct injection into tumors, and methods of reaching cancer cells through vascular injection. Adenovirus (ADV), which has a double-helix gene, has a high gene transfer efficiency and has the advantage of being able to be injected into many cells. However, Adenovirus (ADV) has the disadvantage of being toxic to cells and having an antibody in the body already. In order to compensate for this disadvantage, low molecular chitosan (LMC), PEG (Polyethyleneglycole), and Folate were combined on the surface of the ADV by electrospinning to prepare ADV particles for vascular injection. Electrospinning is a technique for developing nanoscale materials by applying high-pressure electricity to a polymer compound. Chitosan is a natural polymer polysaccharide with excellent biodegradability and biocompatibility. Especially, LMC has hydrophilicity and low cytotoxicity. NH ₂ of LMC forms an ionic bond with COPP of NH ₂ -PEG-COOH, a bifunctional PEG (polyethylene glycol), while simultaneously ionizing crosslinking with tripolyphosphate (TPP). PEG increases solubility and stability and decreases the immune response in the blood stream, allowing for increased residence time. In order to target Folate Receptor (FR) expressed in cancer cells, Folate was able to target cancer cells during vascular injection by amide bond with NH ₂ of PEG.

The aim of this study is to protect ADV from substances in the body, increase survival time, and continuously deliver it to cancer cells to increase delivery efficiency. Thus, the particles were coated with LMC-PEG-Folate (LCPF) ADV to the size capable of vascular injection using electrospinning. LCPF was synthesized by infrared spectroscopy (Furier-Transform Infrared Spectrometer) and hydrogen nuclear magnetic resonance ( ¹ H-Nuclear Magnetic Resonance). As the concentration of LMC increased during electrospinning, the particle size increased with increasing thickness of ADV coating. This was confirmed by DLS and Transmission Electron Microscope. The degree of crosslinking of LMC coated ADV does not increase any more when the concentrations of LMC and TPP increase to a certain level. Based on this, particles were prepared by setting the optimum conditions for electrospinning of LMC and TPP concentrations. Coating efficiency of ADV was determined by quantifying the effective amine groups of LMC by ELISA (Enzyme-linked immunosorbent assay). In addition, the particles were treated with cells to confirm the coating efficiency using a fluorescence microscope (Fluorescence Microscopy, FM) and a fluorescence activated cell sorter (FACS). The effect of Folate molar concentration of ADV-coated particles on LCPF was treated with FR-expressing cells and the results of FM and FACS showed that the FR target effect increased as the molar concentration of Folate increased.

Therefore, it is expected that, through electrospinning, LCPF enables the production of particles for vascular injection and can be used as a carrier to deliver ADV to the desired cancer tissue for the time remaining in the body.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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1 is a schematic diagram of the electrospinning apparatus used in the present invention.

Figure 2 is a graph showing the ζ-potential change of ADV (adenovirus) -cLMC according to LMC (low molecular weight chitosan) concentration.

3 is a TEM image for ADV and crosslinked low molecular weight chitosan (ADV-cLMC). (a), (f) ADV 54.9 ± 0.8 nm, (b), (g) ADV-0.5 wt% cLMC 63.6 ± 1.5 nm, (c), (h) ADV-1.0 wt% cLMC 66.2 ± 2.0 nm, ( d), (i) ADV-1.5 wt% cLMC 67.0 ± 1.4 nm, (e), (j) ADV-2.0 wt% cLMC 68.0 ± 2.0 nm.

4 is a graph showing particle size change with LMC concentration.

5 is a graph showing the change of the effective amino group according to the LMC concentration, the result of chitosan analysis.

6A is an FT-IR graph for PEG-FA (folic acid).

6B is a ¹ H-NMR graph for PEG-FA.

7A is an FT-IR graph for LCPF (LMC-PEG-FA).

7B is a ¹ H-NMR graph for LCPF.

8 is a graph showing particle size of ADV coated with various materials.

9 is a fluorescence microscope image showing the degree of intracellular uptake of ADV-cLMC.

10 is a graph of FACS results showing the degree of intracellular uptake of ADV-cLMC.

FIG. 11A is a fluorescence microscope image showing the degree of intracellular uptake of ADV, ADV-LCP (LMC-PEG) and ADV-LCPF.

11B is a FACS results graph showing the degree of intracellular uptake of ADV, ADV-LCP and ADV-LCPF.

12 is a fluorescence microscope image showing the degree of intracellular uptake of ADV-LCPF according to FA (folic acid) concentration.

13 is a graph of FACS results showing the degree of intracellular uptake of ADV-LCPF according to FA concentration.

Claims (15)

(a) a virus as a gene carrier selected from the group consisting of adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpes simplex virus and basinian virus; And (b) (i) chitosan- (ii) PEG (polyethylene glycol), polyalkylene oxide, polyphenylene oxide, copolymer of PEG and polyalkylene oxide, poly (methoxyethyl) coated on the virus surface Polymers exhibiting anti-biofouling properties selected from the group consisting of methacrylate), poly (methacryloyl phosphatidylcholine), perfluorinated polyethers and dextran- (iii) folic acid, transferrin, vitamin B ₁₂ , vascular endothelial growth factor (VEGF), anti-vascular endothelial growth factor receptor (VEGFR) antibody, anti-HER2 / neu antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-PSA (prostate specific antigen) antibody, anti Biocompatible cancer cell specificity of the virus as a gene carrier comprising a conjugate of cancer cell targeting molecules selected from the group consisting of -PSMA (prostate specific membrane antigen) antibodies and anti-AFP (alpha-fetoprotein) antibodies Half carrier. delete delete delete The carrier vehicle of claim 1, wherein the polymer exhibiting anti-biofouling properties is PEG. delete delete The carrier vehicle of claim 1, wherein the cancer cell targeting molecule is FA (folic acid). delete The delivery vehicle of claim 1, wherein the virus is an adenovirus. The carrier carrier according to claim 1, wherein the chitosan is cross-linked by a crosslinking agent. The carrier of claim 1, wherein the carrier is nanoparticles having a size of 10-500 nm. The transporter of claim 1, wherein the transporter is a transporter for intravenous administration. A pharmaceutical composition for treating cancer, comprising: (a) a pharmaceutically effective amount of the biocompatible cancer cell specific carrier vehicle of any one of claims 1, 5, 8, 10, 11, 12, and 13; And (b) a pharmaceutically acceptable carrier. Composition for cancer imaging, comprising: (a) a pharmaceutically effective amount of the biocompatible cancer cell specific carrier vehicle of any one of claims 1, 5, 8, 10, 11, 12, and 13; And (b) a pharmaceutically acceptable carrier.

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