KR102614058B1 - Reconstituted high-density lipoprotein nanoparticles for drug delivery - Google Patents

Abstract

The present invention relates to reconstituted high density lipoprotein (rHDL) nanoparticles for drug delivery and compositions for preventing or treating cancer containing the same. Specifically, the present invention relates to rHDL nanoparticles based on phospholipid and apolipoprotein containing a drug. The apolipoprotein-based rHDL nanoparticles of the present invention have excellent biological activity.

Description

Reconstructed high-density lipoprotein nanoparticles for drug delivery {RECONSTITUTED HIGH-DENSITY LIPOPROTEIN NANOPARTICLES FOR DRUG DELIVERY}

The present invention relates to reconstituted high density lipoprotein (rHDL) nanoparticles for drug delivery and compositions for preventing or treating cancer containing the same. Specifically, the present invention relates to rHDL nanoparticles based on phospholipid and apolipoprotein containing a drug, and exhibits excellent effects as a drug carrier.

The frequency of occurrence of various diseases is also changing as eating habits and lifestyle habits change. Among them, cancer is the disease that causes the most deaths worldwide. In addition, as of 2019, it is the number one cause of death in Korea (3.6% increase compared to 2018), and the number of cancer cases is steadily increasing as the population ages. In terms of carcinoma and cancer incidence across men and women, the thyroid gland ranked first, followed by the lung, stomach, and colon. Excision, radiation, and chemotherapy are used as treatments. In particular, if the chemotherapy used is not a targeted treatment, the treatment has an effect on general tissues, and drug resistance also exists, so side effects can be reduced by studying the mechanism of anticancer action. There is a need to develop targeted treatments.

The low-density lipoprotein receptor (LDLR, LDL receptor) of cancer cells accepts extracellular cholesterol, a nutrient necessary for growth. And ATP-binding cassette transporter A1 (ABCA1) is involved in major cholesterol efflux and is a factor that increases the accumulation of cholesterol and related derivatives. Therefore, the direction of conducting research on the development of targeted treatments using LDLR and ABCA1, which are closely related to the growth of cancer cells, can be said to be encouraging.

Pediatric central nervous system tumors are the second most common malignant tumor in childhood, and myeloblastoma (MB) is one of the common types. The 5-year survival rate for patients with myeloblastoma is close to 80%, but the treatment regime, which includes resection, radiation, and chemotherapy, has various side effects such as endocrine abnormalities and impaired cognitive function.

Glioma is one of the tumors that frequently occurs in the central nervous system (CNS), accounting for more than 32% of major central nervous system cancers and more than 77% of major malignant brain cancers. Gliomas include glioblastoma (GBM), which exhibits rapid proliferation and angiogenesis characteristics. A variety of new cytotoxic agents have been developed, but they have not improved the average survival rate of patients suffering from glioma due to low blood-brain barrier (BBB) permeability.

A method of delivering drugs to the central nervous system involves the use of polymeric nanoparticles. Numerous studies have reported the ability of polymeric nanoparticles to overcome disruption of the blood-brain barrier and exhibit biological effects. Previous progress has been made to understand the mechanism of nanoparticle entry into brain capillaries, and receptor-related endocytosis is the most likely mechanism.

Doxorubicin hydrochloride (DOX) is used for a variety of tumors. Doxorubicin is commonly used in treatments such as cytotoxic chemotherapy, but may also affect non-malignant tissues. Nevertheless, because it is highly active in malignant tumors, it is used only under close supervision.

Docetaxel (DTX) is used to treat breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, stomach cancer, and prostate cancer. In addition, it is known to induce cell death by inhibiting cancer cell microtubule disassembly and interfering with aggregation.

Temozolomide (TMZ) is used as a treatment for melanoma and brain tumors such as glioblastoma and anaplastic astrocytoma, and has recently shown limited but encouraging activity in patients with metastatic colorectal cancer (mCRC). In addition, it has alkylation/methylation activity at the N-7 or 0-6 position of guanine residues in DNA and acts as an agent that triggers apoptosis.

These anticancer drugs used as standard treatments for cancer have no choice but to be continuously administered in order to show a therapeutic effect, and thus have problems such as drug resistance. In addition, poorly soluble anti-cancer drugs such as docetaxel show toxicity to the body due to the solubilizing agent that dissolves the drug, and do not dissolve well in body moisture, resulting in low bioavailability and treatment efficiency compared to drug administration. Accordingly, in order to increase the cancer target treatment efficiency of anticancer drugs, drug research has been conducted using nanoparticles that increase bioavailability and can pass through the BBB.

The present inventors have conducted various studies to develop a composition that can reduce the in vivo toxicity of anticancer drugs and improve their bioavailability. As a result, various anticancer drugs with different solubilities can be effectively encapsulated in nanoparticles without in vivo toxicity. The present invention was completed by confirming that there is.

Jesse L. et al. Elevating SOX2 levels deleteriously affects the growth of medulloblastoma and glioblastoma cells. 2012, PLOS, 7(8):e44087. Huilong L. et al. Blood-brain barrier modulation to improve glioma drug delivery. 2000, Pharmaceutics, 12(11), 1085. Luigi B. et al. Solid lipid nanoparticle for potential doxorubicin delivery in glioblastoma treatment: preliminary in vitro studies. 2014, Journal of Pharmaceutical Sciences, Vol. 103, 2157-2165. Christopher D. et al. Chemo brain(chemo fog) as a potential side effect of doxorubicin administration: role of cytokine-induced, oxidative/nitrosative stress in cognitive dysfunction. 2010;678:147-56. Jorge E., et al. Docetaxel. 1995, Journal of Clinical Oncology, Vol. 13, 2643-2655. Aida Karachi. et al. Temozolomid for immunomodulation in the treatment of glioblastoma. 2018, Neuro Oncology. Vol. 20, 1566-1572.

The purpose of the present invention is to provide reconstituted high-density lipoprotein (HDL) particles that effectively mimic the biological or physiological role of high-density lipoprotein (HDL) and contain a drug.

The purpose of the present invention is to provide a composition for preventing or treating cancer comprising reconstituted high-density lipoprotein particles containing a drug.

The present invention provides nanoparticles for drug delivery containing one or more of phospholipids, drugs, and apolipoprotein E (eg, Apo E2 and/or Apo E3).

The drugs include docetaxel (molecular weight 808), paclitaxel (molecular weight 854), doxorubicin (molecular weight 580), daunorubicin (527), temozolomide (molecular weight 194), and amrubicin ( amrubicin (molecular weight 483), daunorubicin (molecular weight 528), epirubicin (molecular weight 544), idarubicin (molecular weight 497), mitoxantrone (molecular weight 444), pirarubicin ( pirarubicin (molecular weight 628), bendamustine (molecular weight 358), busulfan (molecular weight 246), carmustine (molecular weight 214), melphalan (molecular weight 305), nimustine (molecular weight) 273), Ranimustine (molecular weight 328), streptozotocin (molecular weight 265), trabectedin (molecular weight 762), vinblastine (molecular weight 811), vincristine (molecular weight 825) ), vindesine (molecular weight 754), and vinorelbine (molecular weight 779), but is not limited thereto.

The drug may have a molecular weight of 1000 or less, and for drugs with a molecular weight of 1000 or more, it is difficult to encapsulate nanoparticles due to the large molecular weight.

In one embodiment, the drug may be temozolomide.

In one embodiment, the drug may be doxorubicin.

In one embodiment, the drug may be docetaxel.

When synthesizing the reconstituted high-density lipoprotein (rHDL) nanoparticles, the synthesis weight ratio of apolipoprotein E and doxorubicin may be 1:1.80 or less, and preferably 1:0.91.

When synthesizing the reconstituted high-density lipoprotein (rHDL) nanoparticles, the synthesis weight ratio of apolipoprotein E and docetaxel may be 1:1.80 or less, and preferably 1:0.91.

When synthesizing the reconstituted high-density lipoprotein (rHDL) nanoparticles, the synthesis weight ratio of apolipoprotein E and temozolomide may be 1:2.20 or less, and preferably 1:1.71.

The phospholipids include 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), and 1,2-dimyristoyl-sn-glycerol. rho-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2- Myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distearoyl-sn-glyce rho-3-phosphocholine (DAPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-diicosanoyl-sn-glycero-3-phos Forcholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidyl Ethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3) -Amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide)(VL-5), dioctadecylamidoglycle spermine 4trifluoroacet Thicic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3dimethylhydroxyethyl ammonium bromide (DORIE), 1,2-dimyristyl Oxy-propyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanami Dium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneammonium bromide (GAP-DLRIE), N-t-butyl- N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl-spermine (GL67) ), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, DLin-DMA. One or more may be selected, preferably DMPC.

The apolipoprotein E may be apolipoprotein E2, E3, or E2 and E3.

In one embodiment, the drug-containing nanoparticles have a diameter of 25 nm or less on the day of synthesis. If the diameter is larger, storage instability increases and it may be difficult to pass the blood-brain barrier.

In one embodiment, the drug encapsulation rate of the drug-containing nanoparticles may be 30% or less.

The nanoparticles of the present invention are high-density lipoprotein (rHDL) nanoparticles made of apolipoprotein and penetrate the blood-brain barrier (BBB) through the low density lipoprotein receptor (LDLR) on the surface. passes through Additionally, because it is translocated into cells through the low-density lipoprotein receptor overexpressed on the surface of brain tumors, more effective treatment is possible compared to drug administration alone. Therefore, it can be used as a suitable carrier for brain tumor treatment.

The present invention provides a composition for preventing or treating cancer containing nanoparticles containing the above drug.

The composition for preventing or treating cancer includes brain tumor, cervical cancer, ovarian cancer, prostate cancer, lung cancer, bile duct cancer, kidney cancer, stomach cancer, liver cancer, retinoblastoma, trophoepithelial cancer, small intestine cancer, colon cancer and rectal cancer, non-small cell lung cancer, and gastric adenocarcinoma. It is a composition for the prevention or treatment of one or more types of cancer selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, bone sarcoma, bladder cancer, and anaplastic astrocytoma, and is preferably applied to brain tumors.

The present invention relates to nanoparticles containing drugs, phospholipids, and apolipoproteins, and compositions containing the same, which have the effect of inducing apoptosis of cancer cells. Therefore, the nanoparticles of the present invention can be used for the prevention or treatment of cancer.

In addition, by using the nanoparticles of the present invention in the drug delivery process, the drug can be transported through the low-density lipoprotein receptor to increase the blood-brain barrier permeability of the drug, which can lead to brain tumors, especially medulloblastoma (MB) and It can increase the treatment efficiency of glioblastoma and reduce systemic side effects through tumor targeting.

Figure 1 is a schematic diagram of a microfluidic device designed to produce nanoparticles using the phospholipid and apolipoprotein E3 of the present invention.
Figure 2 is a dynamic light scattering (DLS) measurement result showing the size distribution of nanoparticles according to intensity according to the synthetic mixture weight ratio of DOX to apolipoprotein E3 when producing nanoparticles containing DOX.
Figure 3 shows the results of dynamic light scattering (DLS) measurement showing the size distribution of nanoparticles according to volume according to the synthetic mixture weight ratio of DOX to apolipoprotein E3 when producing nanoparticles containing DOX.
Figure 4 is a dynamic light scattering (DLS) measurement result showing the size distribution of nanoparticles according to the number of synthetic blended weight ratios of DOX to apolipoprotein E3 when producing nanoparticles containing DOX.
Figure 5 shows the DLS measurement results showing the size distribution of nanoparticles according to intensity according to the synthetic mixture weight ratio of DOX to apolipoprotein E3 6 days after the production of nanoparticles containing DOX.
Figure 6 shows the DLS measurement results showing the size distribution of nanoparticles according to volume according to the synthetic mixture weight ratio of DOX to apolipoprotein E3 6 days after the production of nanoparticles containing DOX.
Figure 7 shows the DLS measurement results showing the size distribution of nanoparticles according to the number of nanoparticles according to the synthetic mixture weight ratio of DOX to apolipoprotein E3 6 days after the production of nanoparticles containing DOX.
Figure 8 is a DLS measurement result showing the size distribution of nanoparticles according to intensity according to the synthetic mixture weight ratio of DTX to apolipoprotein E3 when producing nanoparticles containing DTX.
Figure 9 is a DLS measurement result showing the size distribution of nanoparticles according to volume according to the synthetic weight ratio of DTX to apolipoprotein E3 when producing nanoparticles containing DTX.
Figure 10 is a DLS measurement result showing the size distribution of nanoparticles according to the number of synthetic blended weight ratios of DTX to apolipoprotein E3 when producing nanoparticles containing DTX.
Figure 11 is a DLS measurement result showing the size distribution of nanoparticles according to intensity according to the synthetic mixture weight ratio of DTX to apolipoprotein E3 1 day after manufacturing nanoparticles containing DTX.
Figure 12 is a DLS measurement result showing the size distribution of nanoparticles according to volume according to the synthetic mixture weight ratio of DTX to apolipoprotein E3 1 day after manufacturing nanoparticles containing DTX.
Figure 13 is a DLS measurement result showing the size distribution of nanoparticles according to the number of synthetic blended weight ratios of DTX to apolipoprotein E3 1 day after manufacturing nanoparticles containing DTX.
Figure 14 is a DLS measurement result showing the size distribution of nanoparticles according to intensity according to the synthetic mixture weight ratio of TMZ to apolipoprotein E3 when producing nanoparticles containing TMZ.
Figure 15 is a DLS measurement result showing the size distribution of nanoparticles according to volume according to the synthetic weight ratio of TMZ to apolipoprotein E3 when producing nanoparticles containing TMZ.
Figure 16 is a DLS measurement result showing the size distribution of nanoparticles according to the number of synthetic weight ratios of TMZ to apolipoprotein E3 when producing nanoparticles containing TMZ.
Figure 17 shows the DLS measurement results showing the size distribution of nanoparticles according to intensity according to the synthetic mixture weight ratio of TMZ to apolipoprotein E3 3 days after manufacturing nanoparticles containing TMZ.
Figure 18 shows the DLS measurement results showing the size distribution of nanoparticles according to the volume according to the synthetic mixture weight ratio of TMZ to apolipoprotein E3 3 days after manufacturing nanoparticles containing TMZ.
Figure 19 is a DLS measurement result showing the size distribution of nanoparticles according to the number of synthetic blended weight ratios of TMZ to apolipoprotein E3 3 days after manufacturing nanoparticles containing TMZ.
Figure 20 is an image of the results confirmed through CLSM after treating U87MG cells with DOX solution and DOX-containing nanoparticles for 2 hours to compare the degree of cell entry of DOX solution and DOX-containing nanoparticles through LDLR.
Figure 21 is an image of the results confirmed through CLSM after treating LN229 cells with DOX solution and DOX-containing nanoparticles for 2 hours to compare the degree of cell entry of DOX solution and DOX-containing nanoparticles through LDLR.
Figure 22 is an image of the results confirmed through CLSM after treating Daoy cells with DOX solution and DOX-containing nanoparticles for 2 hours to compare the degree of cell entry of DOX solution and DOX-containing nanoparticles through LDLR.
Figure 23 is a graph regarding the cytotoxicity of DOX solution using U87MG cells.
Figure 24 is a graph regarding the cytotoxicity of DOX solution using LN229 cells.
Figure 25 is a graph regarding the cytotoxicity of DOX solution using Daoy cells.
Figure 26 is a graph regarding the cytotoxicity of DOX-containing nanoparticles using U87MG cells.
Figure 27 is a graph of cytotoxicity of DOX-containing nanoparticles using LN229 cells.
Figure 28 is a graph regarding the cytotoxicity of DOX-containing nanoparticles using Daoy cells.

Hereinafter, with reference to the attached drawings, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present application may be implemented in various forms and is not limited to the embodiments and examples described herein.

Throughout the specification of the present application, when a part "includes" a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

As used herein, the term "reconstituted high density lipoprotein (rHDL)" refers to HDL mimic particles artificially produced to mimic the biological effects of naturally occurring high density lipoprotein (HDL).

The term “apolipoprotein E” refers to a mammalian protein encoded by the APOE gene or a functional variant thereof. In a preferred embodiment, apolipoprotein E is a human protein encoded by the human APOE gene on chromosome 19. Apolipoprotein E may be any of the isoforms of the APOE gene product, such as apolipoprotein E2 (“APOE2”), apolipoprotein E3 (“APOE3”), and apolipoprotein E4 (APOE4). APOE is a polymorphism with three major alleles (epsilon 2, epsilon 3, and epsilon 4). Any allele may be used in various embodiments of the invention. “Functional variant” herein refers to a variant of the mammalian protein encoded by the APOE gene that retains the same or similar biological function as the APOE gene product. In some cases, functional variants include amino acid insertions, deletions, and/or substitutions compared to the protein encoded by the human APOE gene. In some cases, the functional variant is a fragment of the protein encoded by the human APOE gene.

In some embodiments, apolipoprotein E2 is or is at least 95% sequence identical, preferably at least 98% or 99% sequence identical, to the protein disclosed in GenBank under accession number ARQ79459. In some embodiments, apolipoprotein E3 is a protein disclosed in GenBank under accession number ARQ79461.1 or at least 95% sequence identity, preferably at least 98% or 99% sequence identity.

In some embodiments, apolipoprotein E in reconstituted high-density lipoprotein (rHDL) is a recombinant protein produced by genetic engineering, or a synthetic protein produced by chemical synthesis.

The term “apolipoprotein A1” refers to the mammalian protein encoded by the APOA1 gene or functional variants thereof. In a preferred embodiment, apolipoprotein A1 is a human protein encoded by the human APOA1 gene located on chromosome 11. “Functional variant” thereof means a variant of the mammalian protein encoded by the APOA1 gene that retains the same or similar biological function as the APOA1 gene product. In some cases, functional variants include amino acid insertions, deletions, and/or substitutions compared to the protein encoded by the human APOA1 gene. In some cases, the functional variant is a fragment of the protein encoded by the human APOA1 gene.

In some embodiments, apolipoprotein A1 is a protein disclosed in GenBank under accession number AAS68227.1 or at least 95% sequence identity, preferably at least 98% or 99% sequence identity.

In some embodiments, apolipoprotein A1 in reconstituted high-density lipoprotein (rHDL) is a recombinant protein produced by genetic engineering, or a synthetic protein produced by chemical synthesis.

As used herein, the term “apolipoprotein E” is meant to include fragments and functional variants thereof.

As used herein, the term “phospholipid” specifically refers to 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), 1,2 -Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC) ), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2 -Distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-diicosanoyl -sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanol Amine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl) Amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide)(VL-5), dioctadecylamido Glyclespermine 4-trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O- Octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3dimethylhydroxyethyl ammonium bromide (DORIE ), 1,2-dimyristyloxy-propyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N ,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanammonium bromide ( GAP-DLRIE), N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4- Cholesteryl-spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2- It may be one or more selected from the group consisting of DMA and DLin-DMA, but is not limited thereto.

As used herein, the term "doxorubicin" is an anticancer agent, is used for various tumors, and may be referred to as "doxorubicin" or "DOX".

As used herein, the term “docetaxel” is an anticancer agent and is used as a treatment for breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, stomach cancer, and prostate cancer, and may be referred to as “docetaxel” or “DTX.”

As used herein, the term “temozolomide” is an anticancer agent and is used as a treatment for brain tumors such as glioblastoma and anaplastic astrocytoma and melanoma, and may be referred to as “temozolomid” or “TMZ.”

The term “cancer” used herein specifically refers to brain tumor, cervical cancer, ovarian cancer, prostate cancer, lung cancer, bile duct cancer, kidney cancer, stomach cancer, liver cancer, retinoblastoma, trophoepithelial cancer, small intestine cancer, colon and rectal cancer, and non-small cell cancer. It may be one or more selected from the group consisting of lung cancer, gastric adenocarcinoma, acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, bone sarcoma, bladder cancer, and anaplastic astrocytoma, but is not limited thereto.

As used herein, the term "microfluidic device" refers to a device that includes microchannels that allow fluid to flow on a substrate made of various materials including plastic, glass, metal, or silicon, including organic polymer materials. it means.

As used herein, the term "prevention" refers to any act of suppressing or delaying the onset of a disease by administering a composition, and "treatment" means improving or beneficially changing the symptoms of an individual suspected of having a disease or developing a disease by administering a composition. It means all actions that occur.

The present invention will be described in more detail through examples below. However, the examples below are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Production Example 1]

Fabrication of microfluidic devices for nanoparticle production

A microfluidic device as shown in Figure 1 was designed to produce nanoparticles by mixing phospholipids and apolipoproteins. The microfluidic device includes three inlets and one outlet. Of the three inlets, the middle inlet allows hydrophobic phospholipids and drugs, and the two inlets on either side allow It is a structure that injects hydrophilic apolipoprotein E3. In addition, a micropillar structure was introduced inside the microfluidic device to effectively mix lipids and apolipoproteins.

[Production Example 2]

Preparation of doxorubicin-containing nanoparticles

Nanoparticles containing apolipoprotein E3, phospholipid, and doxorubicin were prepared using the microfluidic device as follows.

Doxorubicin (DOX, molecular weight 579.99 g/mol) in DMSO was prepared at a concentration of 10 mg/mL. DMPC solution in absolute ethanol and apolipoprotein E3 solution in PBS were prepared. Then, in one syringe, add the DMPC solution with a concentration of 0.83 mg/mL in about 0.8 mL of absolute ethanol and each of the DOX solutions to the DMPC solution to have concentrations of 1 mg/mL, 2 mg/mL, and 3 mg/mL, respectively. After mixing, the same amount of the apolipoprotein E3 solution was filled into two other syringes, about 1.25 mL each (total about 2.5 mL), and then all syringes were removed from the bubbles.

Each syringe needle was connected to the inlet of the microfluidic device using a tube, and PBS was flowed at an outlet speed of 1 mL/min to clean the microfluidic device. Then, using a syringe pump, the injection flow rate of the DMPC solution was set to 0.8 mL/min and the injection flow rate of the apolipoprotein E3 solution was set to 2.2 mL/min.

The prepared nanoparticles were obtained through the outlet of the microfluidic device, mixed with PBS, and purified twice for 20 minutes at 4°C at maximum centrifuge speed using a 10K filter. After the final purification, approximately 250 μL of DOX-containing nanoparticle solution remained and stored at 4°C.

[Production Example 3]

Preparation of docetaxel-containing nanoparticles

Nanoparticles containing apolipoprotein E3, phospholipids, and drugs were prepared using the microfluidic device in the following manner.

Docetaxel (DTX, molecular weight 807.879 g/mol) in DMSO was prepared at a concentration of 10 mg/mL. DMPC solution in absolute ethanol and apolipoprotein E3 solution in PBS were prepared. Then, in one syringe, add the DMPC solution at a concentration of 0.83 mg/mL in about 0.8 mL of absolute ethanol and each of the DTX to a concentration of 1 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL, respectively. After mixing the solution with the DMPC solution, fill two other syringes, about 1.25 mL each (total about 2.5 mL), with the same amount of the apolipoprotein E3 solution at a concentration of 0.2 mg/mL in PBS, then bubble all syringes. removed.

Each syringe needle was connected to the inlet of the microfluidic device using a tube, and PBS was flowed at an outlet speed of 1 mL/min to clean the microfluidic device. Thereafter, the DMPC solution was injected at a flow rate of 0.8 mL/min and the apolipoprotein E3 solution was injected at a flow rate of 2.2 mL/min using a syringe pump.

The prepared nanoparticles were obtained through the outlet of the microfluidic device, mixed with PBS, and purified twice for 20 minutes at 4°C at maximum centrifuge speed using a 10K filter. After the last purification, approximately 250 μL of a nanoparticle solution containing DTX remained in the residue and stored at 4°C.

[Production Example 4]

Preparation of temozolomide-containing nanoparticles

Nanoparticles containing apolipoprotein E3, phospholipid, and temozolomide were prepared using the microfluidic device as follows.

DMPC solution in absolute ethanol and apolipoprotein E3 solution in PBS were prepared. Then, in one syringe, about 0.8 mL of the DMPC solution at a concentration of 0.83 mg/mL in absolute ethanol was added, and in two other syringes, about 1.25 mL each (total of about 2.5 mL), and the same amount of 0.2 mg/mL in PBS. After filling the apolipoprotein E3 solution, all bubbles were removed from the syringe.

Each syringe needle was connected to the inlet of the microfluidic device using a tube, and PBS was flowed at an outlet speed of 1 mL/min to clean the microfluidic device. Thereafter, the DMPC solution was injected at a flow rate of 0.8 mL/min and the apolipoprotein E3 solution was injected at a flow rate of 2.2 mL/min using a syringe pump.

The prepared nanoparticles were obtained through the outlet of the microfluidic device, mixed with PBS, and purified three times for 20 minutes at 4°C at maximum centrifuge speed using a 10K filter.

Temozolomide (TMZ, molecular weight 194.151 g/mol) in DMSO was prepared at a concentration of 10 mg/mL. Afterwards, it was instilled so that the molar ratio of TMZ to rHDL nanoparticles was 1:100, 1:200, 1:300, and 1:400 and incubated at room temperature for 24 hours. Afterwards, the TMZ-encapsulated nanoparticles were mixed with 2 mL of PBS and purified once for 20 minutes at 4°C at maximum centrifuge speed using a 10K filter. After the final purification, approximately 250 μL of TMZ-encapsulated nanoparticle solution remained and stored at 4°C.

[Example 1]

Optimization of mixing ratio of doxorubicin-containing nanoparticles

To optimize the drug content ratio in anticancer drug-containing nanoparticles, the average size and size distribution of nanoparticles according to the mixing ratio were confirmed using the following method.

The rHDL nanoparticle complex prepared by varying the mixing ratio of doxorubicin and apolipoprotein E3 in the same manner as in Preparation Example 2 was dissolved in PBS, then using Zetasizer Nano ZS, Dynamic Light Scattering (DLS). Changes in size distribution of particles due to agglomeration of nanoparticles were measured, and the results are shown in Table 1 and Figures 2 to 4 below.

On the day of synthesis: based on volume (n=3) ApoE3:DOX
(Synthetic mixture weight ratio) Size (nm) PDI 1:0.91 21.65 ± 1.93 0.41 ± 0.06 1:1.81 15.30 ± 0.27 0.27 ± 0.02 1:2.72 16.13 ± 2.66 0.46 ± 0.04

As shown in Table 1, it can be seen that the DOX-containing nanoparticles of the present invention have a size of 25 nm or less on the day of synthesis.

After storing each nanoparticle at 4°C for 6 days, the particle size was measured and the results are shown in Table 2 and Figures 5 to 7.

After 6 days: Based on volume (n=3) ApoE3:DOX
(Synthetic mixture weight ratio) Size (nm) PDI 1:0.91 23.88 ± 0.00 0.41 ± 0.01 1:1.81 25.57 ± 0.28 0.28 ± 0.00 1:2.72 46.05 ± 0.83 0.83 ± 0.16

As shown in Table 2, when the synthetic weight ratio of DOX to apolipoprotein E3 was 1:1.81 and 1:2.72, instability of the particles was confirmed on the 6th day of synthesis. When the ratio of DOX to apolipoprotein E3 was 1:0.91, the volume increased by 10.30% compared to the day of synthesis, whereas when the synthetic weight ratio was 1:1.81 and 1:2.72, the volume increased by 67.12% and 185.49%, respectively. % increased.

It was confirmed that when the synthetic mixture weight ratio of DOX to apolipoprotein E3 was 1:1.81 or more, more DOX was added than the amount that could be contained in the nanoparticles, and the nanoparticles became unstable due to the uncontained DOX.

Therefore, it is preferable that the synthetic weight ratio of DOX to apolipoprotein E3 is 1:1.80 or less, and therefore, DOX-containing nanoparticles with the same synthetic mixing weight ratio of 1:0.91 were selected.

[Example 2]

Optimization of mixing ratio of docetaxel-containing nanoparticles

The rHDL nanoparticle complex prepared by varying the mixing ratio of DTX and apolipoprotein E3 in the same manner as in Preparation Example 3 above was dissolved in PBS, then using Zetasizer Nano ZS, Dynamic Light Scattering (DLS). Changes in particle size distribution due to the agglomeration of nanoparticles were measured, and the results are shown in Table 3 and Figures 8 to 10.

On the day of synthesis: based on volume (n=3) ApoE3:DTX
(Synthetic mixture weight ratio) Size (nm) PDI 1:0.91 17.66 ± 0.00 0.34 ± 0.14 1:1.82 18.62 ± 0.28 0.28 ± 0.01 1:2.73 21.65 ± 1.93 0.32 ± 0.02 1:3.64 24.06 ± 3.62 0.39 ± 0.04

As shown in Table 3, it can be seen that the DTX-containing nanoparticles of the present invention have a size of 25 nm or less on the day of synthesis.

After storing each nanoparticle at 4°C for 1 day, the particle size was measured, and the results are shown in Table 4 and Figures 11 to 13.

After 1 day: Based on volume (n=3) ApoE3:DTX
(Synthetic mixture weight ratio) Size (nm) PDI 1:0.91 20.69 ± 3.11 0.50 ± 0.02 1:1.82 139.27 ± 11.84 0.53 ± 0.05 1:2.73 125.60 ± 0.00 0.67 ± 0.04 1:3.64 38.71 ± 11.55 0.43 ± 0.01

As shown in Table 4, when the synthetic weight ratio of DTX to apolipoprotein E3 was 1:1.82, 1:2.73, and 1:3.64, instability of the particles was confirmed on the first day of synthesis. When the synthetic blend weight ratio of DTX to apolipoprotein E3 was 1:0.91, the nanoparticle size increased by 17.16% on the first day of synthesis compared to the day of synthesis, whereas the synthetic blend weight ratio of 1:1.82, 1:2.73, and 1:3.64 was increased by 17.16% on the first day of synthesis compared to the day of synthesis. In the case of having it, it increased by 647.96%, 480.14%, and 60.89%, respectively.

It was confirmed that when the synthetic mixture weight ratio of DTX to apolipoprotein E3 was 1:1.82 or more, more DTX was added than could be contained in the nanoparticles, and the nanoparticles became unstable due to the uncontained DTX.

Therefore, it is preferable that the synthetic weight ratio of DTX to apolipoprotein E3 is 1:1.80 or less, and therefore, DTX-containing nanoparticles with the same synthetic mixing weight ratio of 1:0.91 were selected.

[Example 3]

Optimization of mixing ratio of temozolomide-containing nanoparticles

The rHDL nanoparticle complex prepared by varying the mixing ratio of TMZ and apolipoprotein E3 in the same manner as in Preparation Example 4 was dissolved in PBS, and then the size of the particles according to the aggregation of nanoparticles was measured with DLS using Zetasizer Nano ZS. Distribution changes were measured and the results are shown in Table 5 and Figures 14 to 16 below.

On the day of synthesis: based on volume (n=3) ApoE3:TMZ
(Synthetic mixture weight ratio) Size (nm) PDI 1:0.57 15.18 ± 0.00 0.23 ± 0.01 1:1.14 20.69 ± 3.11 0.36 ± 0.05 1:1.71 19.57 ± 1.66 0.39 ± 0.01 1:2.28 29.17 ± 1.00 10.31 ± 0.00

As shown in Table 5, when the synthetic weight ratio of TMZ to apolipoprotein E3 of the present invention is 1:0.57, 1:1.14, and 1:1.71, it is found that it has a size of 25 nm or less on the day of synthesis. You can. However, when the synthetic mixture weight ratio of TMZ to apolipoprotein E3 was 1:2.28 or more, a larger amount of TMZ was added than could be contained in the nanoparticles, and an imbalance in the particles was confirmed due to the drug not contained.

After storing each nanoparticle for 3 days at 4°C, the particle size was measured and shown in Table 6 and Figures 17 to 19 below.

After 3 days: Based on volume (n=3) ApoE3:TMZ
(Synthetic mixture weight ratio) Size (nm) PDI 1:1.14 13.77 ± 1.22 0.34 ± 0.03 1:1.71 17.79 ± 2.68 0.35 ± 0.07

As shown in Table 6, when the synthetic mixture weight ratio of TMZ to apolipoprotein E3 was 1:1.14 and 1:1.71, the nanoparticle size was maintained at a particle size similar to that on the day of synthesis.

Therefore, it is preferable that the synthetic weight ratio of TMZ to apolipoprotein E3 is 1:20 or less, and TMZ-containing nanoparticles with a synthetic weight ratio of TMZ to apolipoprotein E3 of 1:1.71 were selected.

[Example 4]

Composition weight ratio of apolipoprotein E3, phospholipid and drug and doxorubicin encapsulation ratio of doxorubicin-containing nanoparticles

BCA assay, lipid quantification kit, and UV absorption were used to confirm the composition ratio of apolipoprotein E3, phospholipid, and DOX of DOX-containing nanoparticles with a synthetic weight ratio of DOX to apolipoprotein E3 prepared through Preparation Example 2 of 1:0.91. It was quantified using and is shown in Table 7 below.

DOX-containing nanoparticle composition weight ratio Enclosure rate ApoE3:DOX
(Synthetic mixture weight ratio) ApoE3 DMPC DOX DOX (%) 1:0.91 54.41 ± 1.07 44.23 ± 1.04 1.36 ± 0.04 19.3

As shown in Table 7, the composition weight ratio of DOX-containing nanoparticles was confirmed to be 54.41 for apolipoprotein E3, 44.23 for phospholipid, and 1.36 for drug. In addition, it was confirmed that the DOX encapsulation rate was 19.3% when the mixing weight ratio was 1:0.91.

[Example 5]

Composition weight ratio of apolipoprotein E3, phospholipid and drug of docetaxel-containing nanoparticles and docetaxel encapsulation ratio

BCA assay, lipid quantification kit, and HPLC were used to confirm the composition ratio of apolipoprotein E3, phospholipid, and DTX of the DTX-containing nanoparticles having a synthetic weight ratio of DTX to apolipoprotein E3 prepared through Preparation Example 3 of 1:0.91. It was quantified using high-performance liquid chromatography and the results are shown in Table 8 below.

DTX-containing nanoparticle composition weight ratio Enclosure rate ApoE3:DTX
(Synthetic mixture weight ratio) ApoE3 DMPC DTX DTX (%) 1:0.91 57.92 ± 1.10 38.93 ± 2.96 3.15 ± 0.00 16.73

As shown in Table 8, the weight ratio of the DTX-containing nanoparticles was 57.92 for apolipoprotein E3, 38.93 for phospholipid, and 3.15 for drug, and it was confirmed that apolipoprotein E3:DTX had a weight ratio of 1:0.91. In addition, it was confirmed that the DTX inclusion rate was 16.73% when the mixing weight ratio was 1:0.91.

[Example 6]

Composition weight ratio of apolipoprotein E3, phospholipid and drug and temozolomide encapsulation ratio of temozolomide-containing nanoparticles

BCA assay, lipid quantification kit, and HPLC were used to confirm the composition ratio of TMZ, phospholipid, and apolipoprotein E3 in the TMZ-containing nanoparticles having a synthetic weight ratio of TMZ to apolipoprotein E3 prepared through Preparation Example 4 of 1:1.71. It was quantified and the results are shown in Table 9 below.

TMZ-containing nanoparticle composition weight ratio Enclosure rate ApoE3:TMZ
(Synthetic mixture weight ratio) ApoE3 DMPC TMZ TMZ (%) 1:1.71 52.54 ± 1.00 40.37 ± 0.11 7.08 ± 0.02 28.54

As shown in Table 9 above, the composition weight ratio of TMZ-containing nanoparticles was 52.54 for apolipoprotein E3, 40.37 for phospholipid, and 7.08 for drug, and it was confirmed that apolipoprotein E3:TMZ had a weight ratio of 1:1.71. Additionally, when the mixing weight ratio was 1:1.71, the encapsulation rate of TMZ was confirmed to be 28.54%.

[Test Example 1]

Confirmation of cell entry of DOX-containing nanoparticles

The following experiment was performed to confirm cell entry of DOX-containing nanoparticles with a synthetic weight ratio of 1:0.91 of DOX to apolipoprotein E3 prepared through Preparation Example 2.

Cellular uptake of DOX-containing nanoparticles was confirmed in U87MG, LN229, and Daoy cells using confocal laser scanning microscopy (CLSM). U87MG and Daoy cells were cultured using MEM and LN229 cells were cultured using DMEM medium, and 10% FBS and 1% P/S (penicillin-streptomycin) were added to each medium. Each cell was cultured at 37°C in a 5% CO2 atmosphere and 95% relative humidity. Cells were inoculated into an 8-well cell culture slide at 5Х10^4 cells/well, and after 24 hours of culture, medium without FBS and P/S and LDL (low density lipoprotein, human) was prepared as a solution in the medium at a concentration of 50 μg/mL. The prepared medium and LDL solution without FBS and P/S were pre-treated for 2 hours. After 2 hours, the supernatants of the control and experimental groups were removed, and each cell was treated with DOX solution and DOX-containing nanoparticles for 2 hours. After 2 hours, all supernatant was removed, washed with PBS, and cells were fixed with 4% paraformaldehyde (PFA) solution for 10 minutes. Then, 4% PFA solution was removed, mounting medium with DAPI reagent was added, covered with a cover glass, and observed with CLSM. did.

Figures 20 to 22 show whether DOX-containing nanoparticles enter the cells. This corresponds to the results of U87MG cells treated with drugs for 2 hours (Figure 20), LN229 cells treated with drugs for 2 hours (Figure 21), and Daoy cells treated with drugs for 2 hours (Figure 22).

As shown in Figures 20 to 22, more DOX-containing nanoparticles entered the cytoplasm than DOX solution (DOX). To determine whether the nanoparticles of the present invention enter cells through LDLR, cells were pretreated with LDL. Cellular uptake of DOX-containing nanoparticles was inhibited by pretreatment with LDL, a competitive inhibitor for its cognate receptor. Through this, it was confirmed that LDL binds to LDLR and that the nanoparticles of the present invention enter the cells through LDLR.

Therefore, it was confirmed that the nanoparticles of the present invention serve as a drug carrier that effectively delivers DOX to U87MG, LN229, and Daoy cells.

[Test Example 2]

Cytotoxicity test of DOX-containing nanoparticles

Representative glioblastoma cell lines U87MG and LN229 and representative medulloblastoma cell lines Daoy cells were treated with DOX-containing nanoparticles prepared in Preparation Example 2, and cell survival rate was evaluated. Among glioblastoma cell lines, U87MG is known to overexpress LDLR. U87MG, LN229, and Daoy cells were inoculated at 5,000 cells/well in a 96-well cell culture microplate, and after 24 hours of culture, they were treated with DOX solution and DOX-containing nanoparticles at different concentrations from 0 to 20 μg/mL. Cellular toxicity after 24, 48, and 72 hours was measured using absorbance after CCK-8 (Cell Counting Kit-8) treatment, and the results are shown in Table 10 and Figures 23 to 28.

Cell Formulation IC50 values (μM) 24h 48h 72h U87MG DOX solution 9.66 1.51 0.79 DOX-containing nanoparticles 4.93 0.50 0.31 LN229 DOX solution 13.50 3.09 1.58 DOX-containing nanoparticles 12.33 2.57 1.54 Daoy DOX solution 2.03 0.79 0.44 DOX-containing nanoparticles 1.11 0.39 0.34

As shown in Table 10, the DOX solution dissolved in an organic solvent had IC50 values of 9.66 μM (24 hours), 1.51 μM (48 hours), and 792.06 nM (72 hours) in U87MG cells, while the DOX-containing nanoparticles The results showed IC50 values of 4.93 μM (24 hours), 504.31 nM (48 hours), and 311.12 nM (72 hours). Compared to the IC50 of DOX solution, the IC50 of DOX-containing nanoparticles was confirmed to be about 1.96 times lower in 24 hours.

In LN229 cells, DOX solution had IC50 values of 13.50 μM (24 hours), 3.09 μM (48 hours), and 1.58 μM (72 hours), while DOX-containing nanoparticles had IC50 values of 12.33 μM (24 hours) and 2.57 μM (48 hours). ), resulting in an IC50 value of 1.54 μM (72 hours).

In Doay cells, DOX solution had IC50 values of 2.03 μM (24 hours), 786.54 nM (48 hours), and 444.88 nM (72 hours), while DOX-containing nanoparticles had IC50 values of 1.11 μM (24 hours), 393.91 nM (48 hours). ), resulting in an IC50 value of 337.06 nM (72 hours). Compared to the DOX solution IC50, the IC50 of DOX-containing nanoparticles was about 1.83 times lower at 24 hours.

It was confirmed that the DOX-containing nanoparticles of the present invention showed a synergistic effect on toxicity under all test conditions compared to the administration of DOX solution alone.

Claims (20)

Reconstructed high-density lipoprotein (rHDL), which contains phospholipids, apolipoprotein E, and drugs, has a diameter of less than 25 nm, enters cells through LDLR, and is characterized by apolipoprotein E located on the surface. Nanoparticles.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 1, wherein the drug has a molecular weight of 1000 or less.
The method of claim 1, wherein the drug is docetaxel (molecular weight 808), paclitaxel (molecular weight 854), doxorubicin (molecular weight 580), daunorubicin (527), and temozolomide (molecular weight 194). ), amrubicin (molecular weight 483), daunorubicin (molecular weight 528), epirubicin (molecular weight 544), idarubicin (molecular weight 497), mitoxantrone (molecular weight 444) ), pirarubicin (molecular weight 628), bendamustine (molecular weight 358), busulfan (molecular weight 246), carmustine (molecular weight 214), melphalan (molecular weight 305), Nimustine (molecular weight 273), Ranimustine (molecular weight 328), streptozotocin (molecular weight 265), trabectedin (molecular weight 762), vinblastine (molecular weight 811), vin Reconstructed high-density lipoprotein (rHDL) nanoparticles, characterized in that one or more selected from the group consisting of vincristine (molecular weight 825), vindesine (molecular weight 754), and vinorelbine (molecular weight 779).
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 3, wherein the drug is doxorubicin.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 3, wherein the drug is docetaxel.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 3, wherein the drug is temozolomide.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 4, wherein the synthetic weight ratio of apolipoprotein E and doxorubicin is 1:1.80 or less.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 5, wherein the synthetic weight ratio of apolipoprotein E and docetaxel is 1:1.80 or less.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 6, wherein the synthetic weight ratio of apolipoprotein E and temozolomide is 1:2.20 or less.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 7, wherein the synthetic weight ratio of apolipoprotein E and doxorubicin is 1:0.91.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 8, wherein the synthetic weight ratio of apolipoprotein E and docetaxel is 1:0.91.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 9, wherein the synthetic weight ratio of apolipoprotein E and temozolomide is 1:1.71.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 1, wherein the apolipoprotein E is apolipoprotein E2, E3, or E2 and E3.
The method of claim 1, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), 1,2-dimyri Stoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1 -Palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-distea Loyl-sn-glycero-3-phosphocholine (DAPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-diicosanoyl-sn- Glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE) ), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, N1-[2-((1S)-1-[(3-aminopropyl)amino]- 4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide) (VL-5), dioctadecylamidoglyclesper Min 4trifluoroacetic acid (DOGS), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1,2-di-O-octadecenyl -3-Trimethylammonium propane (DOTMA), 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), (1,2-dioleyloxypropyl)-3dimethylhydroxyethyl ammonium bromide (DORIE), 1 ,2-Dimyristyloxy-propyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N- Dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanammonium bromide (GAP-DLRIE) ), Nt-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), ethylphosphocholine (Ethyl PC), dimethyldioctadecylammonium bromide (DDAB), N4-cholesteryl -Spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and D-Lin-MC3-DMA (MC3, DLin-MC3-DMA), DLin-KC2-DMA, DLin -Reconstructed high-density lipoprotein (rHDL) nanoparticles, characterized in that one or more are selected from the group consisting of DMA.
The reconstructed high-density lipoprotein (rHDL) nanoparticle according to claim 1, wherein the drug encapsulation rate is 30% or less.
delete delete A composition for preventing or treating cancer, comprising the reconstructed high-density lipoprotein (rHDL) nanoparticle of any one of claims 1 to 5.
The method of claim 18, wherein the cancer is brain tumor, cervical cancer, ovarian cancer, prostate cancer, lung cancer, bile duct cancer, kidney cancer, stomach cancer, liver cancer, retinoblastoma, trophoepithelial cancer, small intestine cancer, colon cancer and rectal cancer, non-small cell lung cancer, and gastric adenocarcinoma. A composition for preventing or treating cancer, characterized in that it is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, bone sarcoma, bladder cancer, and anaplastic astrocytoma.
The composition for preventing or treating cancer according to claim 19, wherein the cancer is a brain tumor.