HK1193112B - Nanoparticles containing ph-responsive peptide - Google Patents

Abstract

The invention provides nanoparticles capable of releasing a target substance in a weakly acidic pH environment, and a cell transfer agent comprising the same. The nanoparticles comprise a nanoparticle-forming component and a peptide. The nanoparticle-forming component forms a liposome or micelle, and the peptide sequence has 2 to 8 (optionally identical or different) units that begin with His (histidine) and end with an acidic amino acid.

Description

Nanoparticles containing pH-responsive peptides

Technical Field

The present invention relates to a weakly acidic pH-responsive peptide and nanoparticles containing the same. The present invention further relates to a substance-introducing agent containing the nanoparticle.

Background

In cancer chemotherapy, attempts have been made to develop DDS to improve specificity; however, few of these attempts have focused on the tumor environment. Specifically, the tumor tissue is located in a special environment having a pH (pH about 6.5) that is less than the pH under physiological conditions (pH about 7.4). However, drug delivery vehicles must be developed that can function in a tumor tissue specific manner in such a way as to respond to this small pH change. To date, in order to increase blood circulation performance while avoiding binding to plasma proteins in blood, polyethylene glycol (PEG), a hydrophilic macromolecule, has been used to modify the surface of liposomes and the like, and the modified liposomes have been used as carriers of, for example, anticancer drugs (for example, patent literature (PTL) 1). However, PEG has been revealed to be antigenic. Carriers that exhibit PEG on their surface have low affinity for cells and are therefore less likely to be taken up by cells; it is difficult to deliver drugs into tumor cells. The peptide-liposome complex disclosed in PTL2 retains a positive charge due to the presence of a basic amino acid (lysine or arginine) in the N-terminal region, and no charge change occurs depending on pH; and thus sufficient blood circulation performance cannot be expected.

Non-patent literature (NPL)1 uses a His fragment as a pH response region. According to the technique disclosed in NPL1, a sharp decrease in the external ambient pH from 7.4 to 5.0 causes the neutral His to be positively charged, and thus increased electrostatic repulsion causes the destruction of micelles. However, at a weakly acidic pH of 6.5, His alone is not protonated; therefore, it is difficult to cause charge reversal at pH 6.5.

NPL2 discloses a pH-responsive micelle in which the surface charge of the pH-responsive micelle changes from negative to positive when dimethylmaleic acid chemically bound to a lysine moiety at the end of a block polymer is dissociated due to a pH decrease. In the peptide disclosed in NPL2, dissociation of dimethylmaleic acid results in exposure of positively charged lysine residues; this state does not return to the initial state even if the pH is raised. Also, in the case where they pass through an inflammation site or other low pH tissue while flowing in the blood circulation, dimethylmaleic acid is dissociated to expose lysine, causing interaction with blood components; it is difficult to reach the target tumor.

List of cited documents

Patent document

PTL1:JP2004-10481A

PTL2:JP2004-523531A

Non-patent document

NPL1:AIChE Journal Vol.56,No.7,2010,pp.1922-1931

NPL2:International Journal of Pharmaceutics376,2009,pp.134-140

Summary of The Invention

Technical problem

It is an object of the present invention to provide a drug delivery vehicle capable of releasing a target substance in a weakly acidic pH environment such as cancer tissue.

Technical scheme

The present invention provides the following items (1) to (16) relating to a nanoparticle or substance-introducing agent.

(1) Nanoparticles comprising a peptide and a particle-forming component, wherein

The particle-forming component forms liposomes or micelles, and

the peptides have a sequence comprising 2 to 8 units, wherein each unit starts with a His (histidine) and ends with an acidic amino acid, and wherein each unit is the same or different.

(2) The nanoparticle of item (1), wherein each unit has 2 to 5 amino acids between His and an acidic amino acid.

(3) The nanoparticle of item (1) or (2), wherein each unit has 3 amino acids between the His and the acidic amino acid.

(4) The nanoparticle according to item (2) or (3), wherein the amino acid between His and the acidic amino acid is any amino acid selected from the group consisting of: gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, and Asn.

(5) The nanoparticle of item (4), wherein the amino acid between the His and the acidic amino acid is any amino acid selected from the group consisting of: gly, Ala, His, Cys and Ser.

(6) The nanoparticle according to any one of items (1) to (5),

wherein the peptide comprises 2 to 8 units represented by the following formula (I):

His-(AA₁)(AA₂)(AA₃)-Glu/Asp (I)，

wherein His is histidine; Glu/Asp is glutamic acid or aspartic acid; and AA₁、AA₂And AA₃Are identical or different and each denote Gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln or Asn, and

wherein the amino acid sequence of each unit is the same or different.

(7) The nanoparticle of item (6), wherein the peptide has a sequence of any one of SEQ ID Nos:1 to 3.

(8) The nanoparticle of any one of items (1) to (7), wherein the peptide has a hydrophobic group at a terminal end so as to be retained by the liposome or micelle.

(9) The nanoparticle of item (8), wherein the hydrophobic group is C_12-24Hydrocarbyl or C_12-24An acyl group.

(10) The nanoparticle of any of items (1) to (9), wherein the particle-forming component comprises a phospholipid.

(11) The nanoparticle of any of items (1) to (10), wherein the particle-forming component forms a liposome.

(12) The nanoparticle of any of items (1) to (11), wherein the nanoparticle is loaded with at least one target substance selected from the group consisting of: drugs, nucleic acids, peptides, proteins, sugars, and complexes thereof.

(13) A substance-introducing agent comprising the nanoparticle of any one of items (1) to (12).

(14) A peptide compound represented by the following formula (II):

R¹-(Z¹)_l-[His-(AA₁)(AA₂)(AA₃)-Glu/Asp]_n-(Z²)_m-R²(II)，

wherein His is histidine; Glu/Asp is glutamic acid or aspartic acid; AA₁、AA₂And AA₃Are identical or different and each represents Gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln or Asn; n represents an integer of 2 to 8; l and m are the same or different and each represents 0 or 1; r¹Is C_12-24Hydrocarbyl or C_12-24An acyl group; r²Is OH or a C-terminal protecting group; and Z is¹Or Z²Represents a linker arm consisting of 1 to 8 amino acids selected from the following amino acids: gly, Ala, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, and Asn,

the peptide compounds contain a total of 10 to 60 amino acids.

(15) The peptide compound according to item (14), wherein the peptide in the peptide compound represented by the formula (II) has a sequence of any one of SEQ ID NOs:1 to 3.

(16) The peptide compound of item (14) or (15), wherein R¹Is C_12-24An acyl group.

Advantageous effects

The present invention can provide a nanoparticle or substance introducing agent capable of releasing an encapsulated target substance in a weakly acidic cell environment having a pH of about 6.5.

The nanoparticles of the present invention can release a target substance in the above weak acidic region to allow the substance to act therein, and thus can provide an excellent drug delivery system.

Due to the negative charge of the acidic amino acids, the nanoparticles of the invention can avoid interaction with blood plasma components and the like under physiological conditions, i.e., ph7.4, while having long blood circulation properties. In the nanoparticle of the present invention, the presence of an acidic amino acid adjacent to His controls the pH responsiveness of His, so that the nanoparticle of the present invention can exhibit sensitive responsiveness even to a weakly acidic pH. For this reason, after reaching the tumor due to the EPR effect (enhanced permeation and retention effect), the nanoparticles of the invention will be protonated under the weakly acidic conditions in the tumor environment, causing charge reversal; whereby the nanoparticles of the invention are taken up by cancer cells. In view of this, the nanoparticles of the present invention are very useful.

Drawings

FIG. 1 shows the stearylated peptide obtained in production example 1 (SEQ ID NO: 1; C-terminal: CONH)₂) HPLC and MALDI-TOF-MS of (1).

FIG. 2 shows the stearylated peptide obtained in production example 2 (SEQ ID NO: 2; C terminal: CONH)₂) HPLC and MALDI-TOF-MS of (1).

FIG. 3 shows the stearylated peptide obtained in production example 3 (SEQ ID NO: 3; C-terminal: CONH)₂) HPLC and MALDI-TOF-MS of (1).

FIG. 4 shows the stearylated peptide obtained in production example 4 (SEQ ID NO: 4; C terminal: CONH)₂) HPLC and MALDI-TOF-MS of (1).

Fig. 5 shows the results obtained by measuring the amount of intracellular fluorescent dye using a flow cytometer.

Fig. 6 shows the results obtained by observing cells using a confocal laser scanning microscope (Zeiss LSM510 META).

Fig. 7 shows the results obtained by observing nuclei stained with Hoechst33342 (blue) and inclusion bodies/lysosomes stained with LysoTracker Green (Green) using a confocal laser scanning microscope (Zeiss LSM510 META).

Fig. 8 shows the results of the intratumoral kinetic evaluation of the cancer-bearing mice of example 4.

Fig. 9 shows the CD spectra of peptide-only and peptide-modified nanoparticles, and the expected results for secondary structure composition.

FIG. 10 shows the results of cellular uptake of scrambled sequence peptide modified liposomes obtained in production example 4, evaluated using FACS.

FIG. 11 shows the results of cellular uptake of peptide-modified liposomes obtained in production example 2, evaluated using FACS.

Detailed Description

The nanoparticles of the present invention comprise a particle-forming component and a peptide as constituents.

The peptides of the present invention comprise 10 to 60, preferably 12 to 40, and more preferably 14 to 30 amino acids in total.

The peptides of the present invention contain His (histidine, H) and acidic amino acids (glutamic acid (Glu, E) or aspartic acid (Asp, D) as essential constituent elements and have sequences comprising units each starting with His and terminating with an acidic amino acid, each of these units containing 2 to 5, and preferably 3 amino acids between His and an acidic amino acid, each of these units having the same number of amino acids between His and an acidic amino acid, for example, when the initial unit has 3 amino acids, each of the subsequent units also has 3 amino acids, these amino acids are selected from the group consisting of Gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln and Asn. of these amino acids, preferably Gly, Ala, His, Cys and Ser, the peptides have a total of 2 to 8, preferably 2 to 6, and more preferably 2 to 4 units, and adjacent units are directly bonded to each other. Specifically, His is adjacent to acidic amino acids on moieties (moieties) where units directly bind to each other.

In a preferred embodiment, the peptide of the present invention has 2 to 8, preferably 2 to 6, and more preferably 2 to 4 units represented by the following formula (I):

His-(AA₁)(AA₂)(AA₃)-Glu/Asp (I)，

wherein His is histidine; Glu/Asp is glutamic acid or aspartic acid; and AA₁、AA₂And AA₃Each represents Gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, or Asn.

AA₁、AA₂And AA₃Is preferably Gly, Ala, Ser, Cys or His, and more preferably Gly, Ala or His. In the peptide sequence, the total number of His residues is greater than the total number of acidic amino acids. For example, when 2 acidic amino acids are present, 3 to 7 His residues are present. When 3 acidic amino acids are present, 4 to 10 His residues are present. When 4 acidic amino acids are present, 5 to 13 His residues are present. When 5 acidic amino acids are present, 6 to 16 His residues are present. When 6 acidic amino acids are present, 7 to 19 His residues are present. When 8 acidic amino acids are present, 9 to 25 His residues are present. The peptides of the invention contain 2 to 8, preferably 2 to 6, more preferably 2 to 4 acidic amino acids. The peptides of the invention contain 3 to 25, preferably 3 to 19, and more preferably 3 to 13 His residues.

Specifically, the unit of the present invention is preferably His-Gly-Ala-His-Glu, His-Ala-Gly-His-Glu, His-Ala-Ala-Gly-Glu or His-His-Ala-His-Glu. Except thatThese units, the peptide of the present invention may have an amino acid sequence (Z) containing Gly, Ala, His or the like at the N-terminus or C-terminus¹Or Z²(ii) a A connecting arm). Constituting the connecting arm (Z)¹Or Z²) Examples of the amino acid of (a) include: gly, Ala, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, and Asn. Of these amino acids, Gly, Ala, Ser and Cys are preferred; more preferably Gly, Ala and His. Each connecting arm (Z)¹Or Z²) In total, from 1 to 8, preferably from 2 to 6, amino acids are present.

The peptide of the present invention may have a C-terminal protecting group at the C-terminus. The C-terminal protecting group includes a group which can form an amide with a carbon atom of a C-terminal carboxyl group, or a group which can form an ester with an oxygen atom of the carboxyl group. Examples of groups which can form esters include alkyl groups, especially C_1-5Straight or branched alkyl (C)_1-5Alkyl) such as methyl, ethyl and propyl. Examples of groups that can form amides include amine functional groups, such as amino; and alkylamino functional groups, such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and other mono-or di-C_1-5An alkylamino group. Preferably amide-forming groups; more preferably an amino group.

The peptides of the invention are modified with hydrophobic groups. Hydrophobic groups are introduced at the N-or C-terminus, preferably the N-terminus, of the peptide. The hydrophobic group has 12 or more, preferably 12 to 24, more preferably 14 to 22, and still more preferably 16 to 20 carbon atoms. Examples thereof include hydrocarbon groups and acyl groups. Acyl groups are particularly preferred. The hydrophobic group may have a straight chain or a branched chain. Examples of the hydrocarbon group include straight or branched alkyl groups having 12 or more carbon atoms, such as dodecyl, tetradecyl, hexadecyl, octadecyl, and eicosyl. Stearoyl is preferred. Examples of preferred acyl groups include lauroyl, tetradecanoyl, palmitoyl, stearoyl, behenoyloxy, isostearoyl, eicosanoyl, tetracosanoyl, isopalmitoyl, oleoyl, linoleoyl, and the like. More preferred acyl groups are selected from the group consisting of lauroyl, tetradecanoyl, palmitoyl, stearoyl, isostearoyl and oleoyl.

Preferred examples of the peptide of the present invention include peptides having a sequence of any one of amino acid sequences SEQ ID Nos:1 to 3. In a preferred embodiment, the N-terminus of the peptide is bound to a hydrophobic group so as to be retained by the liposome or micelle. Another preferred embodiment is a peptide compound represented by the following formula (II):

R¹-(Z¹)_l-[His-(AA₁)(AA₂)(AA₃)-Glu/Asp]_n-(Z²)_m-R²(II)

wherein His is histidine; Glu/Asp is glutamic acid or aspartic acid; AA₁、AA₂And AA₃Are identical or different and each represents: gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, or Asn; n is an integer of 2 to 8; l and m are the same or different and represent 0 or 1; r¹Is C_12-24Hydrocarbons or C_12-24An acyl group; r²Is OH or a C-terminal protecting group; z¹Or Z²Represents a linker arm consisting of 1 to 8 amino acids selected from the following amino acids: gly, Ala, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, and Asn, and the peptide compound contains 10 to 60 amino acids in total. Examples of the hydrocarbon group, the acyl group and the C-terminal protecting group are as described above.

The peptide of the present invention can be produced by a known peptide synthesis method, particularly a liquid phase synthesis method or a solid phase synthesis method. The peptides of the invention can also be synthesized by a method comprising: the DNA encoding the peptide of the present invention is introduced into a host cell using gene recombination techniques, and the DNA is expressed. For example, in the solid-phase synthesis method, the peptide of the present invention can be obtained as follows: binding an N-terminal protected amino acid carboxyl group to an insoluble resin having an amino group, wherein the amino group of the amino acid corresponding to the C-terminal is protected with a urethane protecting group such as 9-fluorenylmethoxycarbonyl (Fmoc); subsequently removing the protecting group of the amino group to condense the protected amino acids in the N-terminal direction in turn; and removing the protecting groups of the insoluble resin and the amino acid to obtainThe peptide of the invention is obtained. The insoluble resin having an amino group is not particularly limited, but is preferably an Fmoc-NH-SAL resin (4- (2',4' -dimethoxyphenyl-Fmoc-aminoethyl) phenoxy linker resin); the target substance can be directly administered thereto by resin cleavage. The protected amino acids used in the synthesis of the peptides of the invention may be obtained by protecting the functional groups with known protecting groups using known methods. It is also possible to use commercially available protected amino acids. As the protecting group, known protecting groups can be used. Examples thereof include: methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2,2, 2-trichloroethoxycarbonyl, formyl, acetyl, propionyl, butyryl and the like. For the preparation of the protected amino acid, for example, a known method such as DIPCDI-HOBt (diisopropylcarbodiimide-1-hydroxybenzotriazole) method can be used. The condensation reaction may be carried out in a known solvent, for example, an organic solvent such as dimethylformamide. The deprotecting reagent for the amino protecting group is not limited and known reagents such as piperidine/dimethylformamide may be used to cleave the protecting group such as Fmoc. Deprotection of the urethane protecting group may be carried out, for example, by catalytic reduction or using trifluoroacetic acid. Deprotection of other protecting groups may also be carried out by known methods. The degree of progress of the condensation reaction in each synthesis step can be confirmed by a known method such as the ninhydrin reaction method. In view of this, protected peptides having the desired amino acid sequence can be obtained. Using Fmoc-NH-SAL resin as an insoluble resin, the resin and the protecting group can be removed simultaneously by treatment with TMSBr (trimethylsilyl bromide), TFA (trifluoroacetic acid), or the like. COOH (R) may be used depending on the type of resin used²= OH) or CONH₂(R²=NH₂) The C-terminal of (1) to obtain a peptide.

When R is substituted²Defined as OH, the carboxylic acid of the C-terminal amino acid of the peptide of the invention is not replaced. Similarly, when R is substituted²Is defined as NH₂When the carboxyl acid of the C-terminal amino acid of the peptide of the present invention is an amide (CONH)₂)。

The introduction of hydrophobic groups into the peptides of the invention can be accomplished by known methods. For example, the desired acyl group can be introduced by reacting a peptide having a free N-terminus with a carboxylic acid corresponding to the acyl group to be introduced, together with a condensing agent (e.g., HBTU/HOBt) and a reaction accelerator (e.g., DIEA). The introduction of the hydrocarbon group can be effected in the presence of a base by reaction with a halogenated hydrocarbon corresponding to the hydrocarbon group to be introduced.

The thus-obtained peptide of the present invention can be isolated and purified by known methods such as extraction, recrystallization, various chromatographies (gel filtration, ion exchange, partition and adsorption), electrophoresis and counter-current partition. Reversed phase high pressure liquid chromatography is preferred.

At about a neutral pH (e.g., pH7 or 7.4), the nanoparticles of the present invention have a zeta potential of about-100 to 50mV, preferably about-50 to 30mV, more preferably about-30 to 10mV, and especially-30 to 0 mV. The zeta potential can be measured by using a zeta potential analyzer (Zetasizer).

Although it is not limited thereto, the nanoparticles of the present invention have, for example, an average particle diameter of 30 to 1000nm, preferably 50 to 500nm, more preferably 60 to 400nm, and particularly 70 to 300 nm. For example, the average particle diameter can be measured by a dynamic light scattering method, a static light scattering method, electron microscope observation, atomic force microscope observation, or the like.

The substance-introducing agent of the present invention can be used in vitro or in vivo to deliver a target substance to a low pH site.

Examples of low pH sites include sites of inflammation, tumors, sites of infection, and the like. Tumor sites are particularly preferred.

Examples of target species that may be loaded within nanoparticles include, but are not particularly limited to, one or more members selected from the group consisting of: drugs, nucleic acids, peptides (e.g., oxytocin, bradykinin, thyroid stimulating hormone releasing factor, enkephalin and possibly bioactive peptides and peptide hormones), proteins (e.g., enzymes, interleukins and various possible cytokines, cell transfer factors, cell growth factors and antibodies), sugars and combinations thereof. These substances may be selected according to the purpose, such as diagnostic or therapeutic purposes. Nucleic acids include DNA and RNA, as well as analogs and derivatives of DNA and RNA (e.g., siRNA, Peptide Nucleic Acids (PNA), and thiophosphonic acid DNA). The nucleic acid may be single-stranded or double-stranded, and may be a linear or cyclic nucleic acid.

Examples of drugs that can be used as the target substance include: anti-cancer drugs, vasodilators, antimicrobial agents, and the like. Specific examples of the anticancer drugs include: tegafur, adriamycin, daunomycin, cisplatin, oxaliplatin, carboplatin, paclitaxel, irinotecan, SN-38, dactinomycin, vincristine, vinblastine, methotrexate, azathioprine, fluorouracil, mitomycin C, docetaxel, cyclophosphamide, capecitabine, epirubicin, gemcitabine, mitoxantrone, folinic acid, vinorelbine, trastuzumab, etoposide, estramustine, prednisone, interferon alpha, interleukin-2, bleomycin, ifosfamide, mesna, hexamethylmelamine, topotecan, cytarabine, methylprednisolone, dexamethasone, mercaptopurine, thioguanine, fludarabine, gemtuzumab, idarubicin, mitoxantrone, retinoic acid, alemtuzumab, cladribine, imatinib, epirubicin, dacarbazine, procarbazine, dactinomycin, paclitaxel, and the like, Nitrogen mustard, rituximab, dinierein diftotox, trimethoprim/sulfamethoxazole, allopurinol, carmustine, tamoxifen, filgrastim, temozolomide, melphalan, vinorelbine, azacitidine, Thalidomide (thiaidomide), mitomycin, and the like. Examples of vasodilators include: bosentan, ambrisentan, sodium prostaglandins, and the like. Examples of antimicrobial agents include: amphotericin B, penicillin G, ampicillin, cefazolin, imipenem, aztreonam, gentamicin, tetracycline, chloramphenicol, erythromycin, azithromycin, rotamycin, telithromycin, quinupristin, fosmidoxin (phospine), nalidixic acid, norfloxacin, sparfloxacin, linezolid, and the like.

Preferred examples of nucleic acids that can be used as target substances include any double stranded RNAs (dsRNAs) selected from the group consisting of: meroduplex RNA (mdRNA), nicked dsRNA (ndsrna), gapped dsRNA (gdrna), short interfering nucleic acid (siNA), siRNA, small RNA (mirna), short hairpin RNA (shrna), short interfering oligonucleotides, short interfering replacement oligonucleotides, short interfering modified oligonucleotides, chemically modified dsRNA, and post-transcriptional gene silencing RNA (ptgsrna). The target substance may be used alone or in combination of two or more. For example, two or more types of siRNAs may be used in combination.

In one embodiment of the substitution and modification (including chemical modification) aspects, the double stranded RNA may comprise a 1 to 4 nucleotide overhang at one or both 3' ends of the double stranded RNA, such as an overhang comprising one deoxynucleotide or two deoxynucleotides (e.g., thymidine, adenine). The double-stranded RNA may have a blunt end at one or both ends of the double-stranded RNA. In one embodiment, the 5' end of the first or second strand is phosphorylated. In any embodiment of the double-stranded RNA, the nucleotide overhang at the 3' end can comprise ribonucleotides or deoxyribonucleotides that are chemically modified on the sugar, base, or backbone of the nucleic acid. In any embodiment of the double stranded RNA, the nucleotide overhang at the 3' end may comprise one or more universal basic nucleotides. In any embodiment of the double stranded RNA, the nucleotide overhang at the 3' end may comprise one or more acyclic nucleotides. In any embodiment of the double stranded RNA, the dsRNA may further comprise a terminal phosphate group, such as a 5' -phosphate (see Martinez et al, cell.110:563-574,2002 and Schwarz et al, Molec.cell.10:537-568,2002) or a 5',3' -diphosphate.

The double stranded RNA may further comprise 2' -sugar substitutions, such as 2' -deoxy, 2' -O-methyl, 2' -O-methoxyethyl, 2' -O-2-methoxyethyl, halogen, 2' -fluoro, 2' -O-allyl, or the like, or combinations thereof. In other embodiments, the double stranded RNA further comprises a terminal cap substitution, such as an alkyl, abasic, deoxyabasic, propanetriyl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or a combination thereof, on one or both ends of the first strand or on one or more of the second strands.

In other embodiments, the double stranded RNA can further comprise at least one modified internucleotide linkage, such as independently, a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methylphosphonate, alkylphosphonate, 3' -alkylenephosphonate, 5' -alkylenephosphonate, chiral phosphonate, phosphonoacetate, phosphonothioacetate, phosphinate, phosphoramidate, 3' -phosphoramidate, aminoalkyl phosphoramidate, thiocarbonyl alkylphosphonate, phosphorothioyl phosphotriester, phosphoroselenoate, phosphorborane linkage, or a combination thereof.

Double-stranded RNA can be replaced or modified (including chemically modified) by using: 5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl, 2-propyl or other alkyl derivatives of adenine and guanine; 8-substituted adenine and guanine (e.g., 8-aza, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy); 7-methyl, 7-deaza and 3-deaza adenine and guanine; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (e.g., 5-bromo or 5-fluoro), 5-trifluoromethyl or other 5-substituted uracils and cytosines; and nucleotide analogs such as 6-azauracil.

RNAs, such as double stranded RNAs (dsRNAs), may be chemically modified. Examples of such chemical modifications include, but are not limited to: phosphorothioate internucleotide linkages, 2' -deoxyribonucleotides, 2' -O-methyl ribonucleotides, 2' -deoxy-2 ' -fluoro ribonucleotides, "acyclic" nucleotides, 5' -C-methyl nucleotides and terminal triosyl and/or inverted deoxyabasic residues. These chemical modifications can protect RNAi activity in cells.

The liposome may be a Multilamellar Liposome (MLV) or a unilamellar liposome, such as SUV (small unilamellar vesicles), LUV (large unilamellar vesicles) or GUV (large unilamellar vesicles), as long as it is a closed vesicle having a lipid bilayer structure.

Specific examples of the types of lipids that can form a lipid bilayer within the liposomes of the present invention include: lecithins (e.g., dioleoyl lecithin, dilauroyl lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, and distearoyl lecithin), phosphatidylglycerols (e.g., dioleoyl phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and distearoyl phosphatidylglycerol), phosphatidylethanolamines (e.g., dioleoyl phosphatidylethanolamine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine), phosphatidylserines, phosphatidylinoses, phosphatidic acids, cardiolipins, and possibly phospholipids or hydrogen adducts thereof; and sphingomyelin, gangliosides and possibly glycolipids. These lipids may be used alone or in combination of two or more. The phospholipid may be a synthetic lipid, a semi-synthetic lipid, or a natural lipid derived from egg yolk, soy, or other animal or plant sources (e.g., egg yolk lecithin and soy lecithin). These lipids may be used alone or in combination of two or more.

In order to obtain physical or chemical stability of the lipid bilayer and to adjust its membrane fluidity, the lipid bilayer may comprise one or more members selected from the group consisting of, for example: cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, dehydrocholesterol, dihydrocholesterol and possibly sterols of animal origin; stigmasterol, sitosterol, campesterol, brassicasterol and possibly sterols of plant origin (phytosterols); zymosterol, ergosterol and possibly sterols of microbial origin; glycerol, sucrose and possibly sugars; triolein, tricaprylin and possibly glycerol fatty acid esters. Their amount is not particularly limited, but is preferably 5% to 40% (molar ratio), more preferably 10% to 30% (molar ratio), based on the total amount of lipids constituting the bilayer.

The lipid bilayer may comprise: tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and possibly an antioxidant; stearamides, oleamides and possibly charged materials for providing a positive charge; dicetyl phosphate and possibly a charged material for providing a negative charge; membrane-external proteins, membrane-internal proteins and possibly membrane proteins. Their amounts can be appropriately adjusted.

The nanoparticle of the present invention comprises a peptide comprising 10 to 60 amino acids on its surface. When the surface of the nanoparticle is modified with the peptide of the present invention, it is preferable to modify about 1 to 10mol% of the total lipid constituting the nanoparticle. The liposome surface of the unilamellar liposomes is the outer surface of the lipid bilayer, while the liposome surface of the multilamellar liposomes is the outer surface of the outermost lipid bilayer. The nanoparticle of the present invention may comprise the aforementioned peptide at a position other than the surface (e.g., the inner surface of a lipid bilayer).

The nanoparticles of the invention preferably comprise a helper lipid (helper lipid). Examples of helper lipids include EPC (egg lecithin), DLPC (dilinoleoyl lecithin), DMPC (dimyristoyl lecithin), DPPC (dipalmitoyl lecithin), DSPC (distearoyl lecithin), POPC (palmitoyl oleoyl lecithin), DOPC (dioleoyl lecithin), DOPE (dioleoyl phosphatidylethanolamine), SOPE (stearoyl oleoyl lecithin), and the like. Of these lipids, EPC, DOPC, DOPE and SOPE are preferred.

An illustrative example of the production of liposomes using the hydration method is described below.

The lipid as a constituent component of the lipid bilayer and the aforementioned peptide modified with a hydrophobic group or a hydrophobic compound are dissolved in an organic solvent, followed by removal of the organic solvent by evaporation, thereby obtaining a lipid film. Examples of the organic solvent used herein include: hydrocarbons such as pentane, hexane, heptane and cyclohexane; halogenated hydrocarbons such as methylene chloride and chloroform; aromatic hydrocarbons such as benzene and toluene; lower alcohols such as methanol and ethanol; esters, such as methyl acetate and ethyl acetate; ketones, such as acetone; and so on. These organic solvents may be used alone or in combination of two or more. Subsequently, the lipid film is hydrated and stirred or sonicated, thereby producing nanoparticles having the aforementioned peptides on their surfaces.

Micelles can be prepared by using only the peptide of the present invention containing a hydrophobic group such as stearoyl (preferably acyl). In this case, the peptide also serves as a particle-forming component. Micelles can also be prepared by using the peptides of the invention in combination with other components such as phospholipids or surfactants that can form micelles.

As the phospholipid, the above-listed liposome-forming phospholipids and auxiliary lipids can be used. As the surfactant (anion, nonionic and cation), the following can be used.

Examples of the anionic surfactant include: sulfonates such as alkylsulfonates, paraffin sulfonates, alkylbenzenesulfonates, α -olefin sulfonates, sulfosuccinates and sulfosuccinates (e.g., dioctyl sodium and disodium laureth sulfosuccinate), isethionates, acyl isethionates (e.g., sodium 2-lauroyloxyethane sulfonate) and sulfoalkylamides of fatty acids, especially N-acylmethyltaurines; sulfates such as alkyl sulfates, ethoxylated alkyl sulfates, sulfated monoglycerides, sulfated alkanolamides, and sulfated fats and oils; carboxylates, such as alkyl carboxylates having a carbon chain length of 12 or more carbon atoms, acyl sarcosinates, sarcosinates (e.g., sodium dodecyl sarcosinate), sodium ethoxy carboxylates, carboxylic acids and salts (e.g., potassium oleate and laurate), ether carboxylic acids; ethoxy carboxylic acids and salts, such as sodium carboxymethyl alkyl ethoxylate; phosphates and salts (e.g., lecithin); acylglutamates (e.g., disodium n-lauroyl glutamate) and mixtures thereof.

Examples of the nonionic surfactant include: polyoxyethylene such as ethoxylated fatty alcohols, ethoxylated alcohols (e.g., octyloxyethylene ethylene glycol monocetyl ether, C16E8, and C12E8), ethoxylated fatty acids, ethoxylated fatty amines, ethoxylated amides, ethoxylated alkanolamides, and ethoxylated alkylphenols; phosphoric acid triesters (e.g., sodium dioleyl phosphate); an alkylamide diethylamine; alkylamidopropyl betaines (e.g., cocamidopropyl betaine); amine oxide derivatives such as alkyldimethylamine oxide, alkyldihydroxyethylamine oxide, alkylamidodimethylamine oxide, and alkylamidodihydroxyethylamine oxide; polyhydroxy derivatives such as polyol esters and ethers (e.g., sucrose monooleate, cetostearyl glucoside, β -octyl furanoglucoside, alkyl glucosides having carbon chain lengths of 10 to 16 carbon atoms), mono-, di-and polyglyceryl ethers and polyglycerol esters (e.g., tetraglycerol monolaurate and tetraglycerol and triglycerol monooleates (e.g., TS-T122 produced by Grinsted), diglycerol monooleate (e.g., TST-T101 produced by Grinsted)) and ethoxylated glycerides; monoglycerides, such as oleic acid monoglyceride and linoleic acid monoglyceride; and diglyceride fatty acids such as diglyceride monoisostearate.

Examples of cationic surfactants include: aliphatic-aromatic quaternary ammonium halides; quaternary ammonium alkylamide derivatives; alkylamidopropyl dimethyl ammonium lactate; alkylamidopropyl dihydroxyethyl ammonium lactate; alkyl amide propyl morpholine lactate, and the like.

The nanoparticle in liposome form of the present invention can be prepared as follows.

Lipids, which are constituent components of lipid bilayers, are dissolved in an organic solvent, and the organic solvent is subsequently removed by evaporation, thereby obtaining a lipid film. The lipid film is hydrated and stirred or sonicated to produce nanoparticles. Subsequently, the aforementioned peptide modified with a hydrophobic group or a hydrophobic compound is added to the external liquid of the nanoparticle. So that the peptide can be introduced onto the surface of each nanoparticle.

In preparing the nanoparticles, the ratio of cationic lipid (EtOH solution)/helper lipid (EtOH solution)/Chol (EtOH solution) may be appropriately changed. When PEG is used for the modification, the ratio of PEG is appropriately adjusted. For example, PEG may be added in an amount of 0.1 to 15mol% based on the total amount of lipids.

The nanoparticles of the present invention may encapsulate a substance of interest to be delivered into a cell.

When the target substance is water-soluble, it is added to the aqueous solvent used in making the lipid film hydrate during the preparation of the nanoparticles; the target substance can thus be encapsulated in the aqueous phase of the nanoparticles. When the lipid solubility is achieved, the target substance is added into an organic solvent used for preparing the nano particles; the target substance can thus be encapsulated in the lipid bilayer of the nanoparticle. As used herein, the term "package" refers to both of the following: the target substance is contained within a hollow particle, such as a nanoparticle, and the target substance is carried on a surface as a carrier, such as a lipid bilayer. The biological species to which the target substance is delivered is not limited as long as it is a vertebrate. Mammals are preferred. Examples of mammals include: humans, apes, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like.

The nanoparticles of the present invention may be used in a dispersed state. As the dispersion solvent, a buffer solution such as a physiological saline solution, a phosphate buffer solution, a citrate buffer solution, or an acetic acid buffer solution may be used. For dispersion, additives such as saccharides, polyols, water-soluble polymers, nonionic surfactants, antioxidants, pH adjusters and hydration promoters may be added.

The nanoparticles of the invention may also be used in a dry dispersion (e.g., freeze-dried or spray-dried). The dispersion state may be prepared by adding the dried nanoparticles to a buffer solution such as a physiological saline solution, a phosphate buffer solution, a citrate buffer solution, or an acetic acid buffer solution.

Nanoparticles can be used in vitro and in vivo. When the nanoparticles are used in vivo, the route of administration may be, for example, intravenous injection, intravenous drip, or the like. The dosage and administration frequency can be appropriately adjusted according to the type and amount of the target substance encapsulated in each nanoparticle of the present invention.

The nanoparticles of the present invention cause neither weight loss nor liver disease, and thus can be safely administered.

Examples

The present invention is described in more detail below with reference to production examples and examples. However, the scope of the present invention is not limited to these examples.

Production example 1

Synthesis of stearoylated peptide (Compound 1)

Compound 1: c₁₇H₃₅-C(O)-GGGGHGAHEHAGHEHAAGEHHAHE-NH₂

Synthesis of SEQ ID NO:1 (C-terminus: CONH) by Fmoc solid phase Synthesis on a 0.1mM or 0.03mM scale using Rink amide resin (0.67mMol/g) as starting Material, using amino acids, condensing agent (HBTU/HOBt) and reaction Accelerator (DIEA) (4 equivalents each relative to resin)₂) The peptide of (1). Stearic acid (m.w.284.48), a condensing agent (HBTU/HOBt), and a reaction accelerator (DIEA) (4 equivalents each with respect to the resin) were added to the resin to cause activation, and then the resultant product was added to the resin in such a state and reacted at room temperature overnight: where the extension of the amino acid has been completed leaving only the N-terminus free. (HBTU: M.W.379.2; HOBt: anhydrous, M.W.135.1; DIEA: M.W.129.2). After completion of the reaction, a mixture solution of TFA (trifluoroacetic acid) (TFA: 125 mL; H)₂O: 0.25 mL; phenol: 0.375 g; ethanedithiol: 0.125 mL; and thioanisole: 0.25mL) was added to the resin, and reacted for 15min under ice-cooling at room temperature for 2h to obtain a crude peptide. Purification was performed by HPLC followed by lyophilization. Purity was measured by HPLC and MALDI-TOF-MS. The analysis was performed under the following HPLC conditions, and the target product was obtained as a single peak (retention time 15.1 min).

Buffer A: 0.1% TFA/H₂O; b, buffer solution: 0.1% TFA/acetonitrile; column: SunFire C18 column, 5 μm, 4.6x150 mm; flow ofFast: 1 mL/min; wavelength: 220 nm.

The Voyager system from Applied Biosystems was used for MALDI-TOF-MS. Calculated molecular weight: 2651.8, respectively; molecular weight found: 2651.73. FIG. 1 shows the resulting synthesis scale of HPLC and MALDI-TOF-MS: 0.1-mM scale (molecular weight: 2651.8); amount of resin used: 159.8 mg; theoretical values of the peptides obtained by using this resin: 283.9 mg; the crude yield actually obtained: 183.1mg (yield: 64.5%).

Production example 2

Synthesis of peptide with reduced 4 AA (Compound 2)

Compound 2: C₁₇H₃₅-C(O)-GGGGHGAHEHAGHEHAAGEH-NH₂

The synthesis was performed as described in production example 1 to obtain the target peptide. The target peptide contains a stearoyl group (stearic acid amide) at the N-terminus and a CONH at the C-terminus₂. The peptide has the amino acid sequence of SEQ ID NO 2.

Calculated molecular weight of target stearoylated peptide: 2177.3, respectively; molecular weight found: 2177.9. FIG. 2 shows the results of HPLC and MALDI-TOF-MS.

The synthesis scale is as follows: 0.03-mM scale (molecular weight: 2177.3); amount of resin used: 61.2 mg; theoretical values of the peptides obtained by using this resin: 89.3 mg; the crude yield actually obtained: 28.3mg (31.7% yield).

Production example 3

Synthesis of peptide with reduced 8 AA (Compound 3)

Compound 3: c₁₇H₃₅-C(O)-GGGGHGAHEHAGHEHA-NH₂

The synthesis was performed as described in production example 1 to obtain the target peptide. The target peptide contains a stearoyl group (stearic acid amide) at the N-terminus and a CONH at the C-terminus₂. The peptide has the amino acid sequence of SEQ ID NO. 3.

Calculated molecular weight of target stearoylated peptide: 1782.9, respectively; molecular weight found: 1782.4. FIG. 3 shows the results of HPLC and MALDI-TOF-MS.

The synthesis scale is as follows: 0.03-mM scale (molecular weight: 1782.9); amount of resin used: 67.0 mg; theoretical values of the peptides obtained by using this resin: 80.0 mg; the crude yield actually obtained: 51.4mg (yield: 64.3%).

Production example 4

Synthesis of scrambled peptide (comparative Compound)

Compound 4: c₁₇H₃₅-C(O)-GGGGHGEAHHAEGHHAEAHHGEAH-NH₂

The synthesis was performed as described in production example 1 to obtain the target peptide. The target peptide contains a stearoyl group (stearic acid amide) at the N-terminus and a CONH at the C-terminus₂. The peptide has the amino acid sequence of SEQ ID NO. 4.

Calculated molecular weight of target stearoylated peptide: 2651.8, respectively; molecular weight found: 2651.0. FIG. 4 shows the results of HPLC and MALDI-TOF-MS.

The synthesis scale is as follows: 0.03-mM scale (molecular weight: 2651.8); amount of resin used: 45.3 mg; theoretical values of the peptides obtained by using this resin: 80.5 mg; the crude yield actually obtained: 31.0mg (yield: 38.5%).

Example 1: measurement results of particle diameter and surface potential (zeta-potential) under different pH conditions (showing pH responsiveness)

(1) Liposomes were prepared as follows. Specifically, a mixture lipid ethanol solution prepared by mixing egg yolk lecithin (EPC) and tetraoleoyl diammonium propane (DOTAP) at a ratio of 8:1 (mol ratio) was distributed in a test tube and mixed therewith with an equal amount of chloroform, followed by evaporation drying under a nitrogen stream to obtain a thin lipid film. A buffer solution of pH7.4 was added thereto, and the mixture was fully hydrated at room temperature for 10 min. After completion of the hydration, the test tube was sonicated using a water tank type sonication apparatus to prepare liposomes (lipid concentration: 10 mM). To the resulting liposome suspension was added the peptide obtained in production example 1 (compound 1) in an amount of 5mol% of the total lipid content, and the mixture was incubated. Electrostatic interactions cause the peptide to bind to the surface of the lipid membrane and move the stearoyl group of the peptide to (intercalate into) the hydrophobic portion of the membrane lipids. Thus preparing liposome 1 with the surface modified by peptide.

(2) The particle size (size) and surface potential (zeta potential) of liposomes 1 diluted and suspended in buffer solutions with different pH were measured by Zetasizer Nano potential analyzer manufactured by Malvern Instruments ltd. Table 1 shows the results.

TABLE 1

When the pH is 7.4, the particle size is slightly less than 200 nm. Even when the pH value was decreased, no large change in particle size was observed. The surface potential was about-15 mV at pH 7.4; however, when the pH was 6.5, the surface potential increased to 7 mV. This indicates that the surface charge changed from negative to positive due to a slight change in pH.

Example 2: measurement of cellular uptake at different pH conditions (showing pH responsiveness)

Liposomes were prepared by the preparation method in example 1 except that a fluorescent dye-labeled lipid (rhodamine-labeled dioleoylphosphatidylethanolamine) was previously added to the lipid solution in an amount of 1mol% of the lipid content. The prepared relevant liposomes were added to mouse melanoma cells (B16-F1) cultured in media with different pH (5.5, 6.0, 6.5 and 7.4) and cultured at 37 ℃ for 1 hour. Thereafter, the culture supernatant was removed therefrom, and the cells were harvested by trypsinization. The amount of fluorescent dye in the cells (the amount of uptake of the liposomes by the cells) was measured by flow cytometry (FACS Caliber flow cytometer). The results are shown in FIG. 5.

At pH7.4, the amount of liposomes involved in cellular uptake was almost the same as that of untreated cells (no liposomes added). In contrast, when the pH is 5.5 or 6.5, a large amount of the relevant liposome is taken up by the cells. In particular, by slightly changing the pH (from 7.4 to 6.5), the uptake of relevant liposomes by the cells was significantly increased.

Cells were observed here using a confocal laser scanning microscope (Zeiss LSM510 META). The results are shown in FIG. 6. At pH7.4, little fluorescence of the relevant liposomes was observed intracellularly (red color). In contrast, when the pH was 6.5 or 5.5, a large amount of fluorescence of the relevant liposome was observed in the cytoplasm (blue is a nucleus stained with Hoechst 33342). This also shows that by slightly changing the pH (from 7.4 to 6.5) the amount of relevant liposomes taken up by the cells is significantly increased.

Example 3: observation of intracellular kinetics in cultured cell systems (display of pH responsiveness in Inclusion bodies)

As described in example 2, fluorescently labeled relevant liposomes (red) were added to mouse melanoma cells (B16-F1) cultured in media with different pH (5.5, 6.0, 6.5, and 7.4) and cultured for 1 hour at 37 ℃. Thereafter, nuclei were stained with Hoechst33342 (blue), while inclusion bodies and lysosomes were stained with LysoTracker Green (Green). Observations were then made using a confocal laser scanning microscope (Zeiss LSM510 META). The results are shown in FIG. 7.

The results were similar to those of example 2; specifically, at pH7.4, little red coloration was observed intracellularly, indicating that the relevant liposomes were not taken up by the cells. In contrast, when the pH was 6.5 or 5.5, a large amount of red coloration was observed inside the cells, indicating that many of the relevant liposomes were taken up by the cells. The red coloration does not overlap with the green coloration; almost all red coloration is only red. This means that the relevant liposomes are not retained in the inclusion body or lysosomes, but escape into the cytoplasm. The inclusion bodies and lysosomes have a low pH environment within them. In view of this, it was suggested that there is a possibility that the relevant liposome is altered to fuse with, for example, an endosomal membrane or lysosome membrane, and thereby escape from the endosome or lysosome. This demonstrates the ability of the relevant liposomes to escape from inclusion bodies and lysosomes in response to changes in pH within the inclusion bodies and lysosomes.

Example 4: kinetics of tumor in cancer bearing mice (display of pH responsiveness within tumor)

0.2mL of relevant liposomes containing a fluorescent dye (CellTracker CM-DiI (Red)) at a concentration of 0.5% lipid content (lipid concentration: 10mM), or as a control 0.2mL of polyethylene glycol (PEG) -modified liposomes containing CellTracker CM-DiI similar to the above at a concentration of 0.5% lipid content (lipid composition: EPC: cholesterol: PEG 2000-modified distearoylphosphatidylethanolamine =1.85:1: 0.15; lipid concentration: 10mM), was intravenously injected via the tail vein into a patient with a growth to 100mM formed by subcutaneous transplantation of B16-F1 cells³Cancer hairless mice with large and small tumors. Thereafter, the tumors were excised and cryo-segmented. The frozen sections were treated with 4% paraformaldehyde to fix the tissues, followed by treatment with an anti-CD 31 antibody (an antibody against endothelial cell marker protein) as a primary antibody. Subsequently, additional treatment was performed using a fluorochrome (Alexa488 (green)) labeled antibody as a secondary antibody to immunolabel the fixed tissue. In addition, the same fixed tissue was embedded (embedded) in Vectashield encapsulation tablets containing nuclear Dye (DAPI). The embedded and fixed tissues thus obtained were observed with a confocal laser scanning microscope. Table 8 shows the results.

Two liposomes (red) were observed in equal amounts in tumor tissue. This indicates that the relevant liposomes have substantially the same long blood circulation properties as PEG, although the relevant liposomes are not coated with PEG. In addition, many PEG liposomes (red) were observed together with green (yellow); this indicates that the PEG liposomes are placed in and around the blood vessels. Thus, it was hypothesized that PEG liposomes might be leaked from tumor tissue. In contrast, with respect to the relevant liposomes, the red color alone was observed to be present far from the green color, indicating that the relevant liposomes are located far from the blood vessels, i.e., deep within the tumor tissue. This suggests that the relevant liposomes may remain in the tumor (with excellent targeting effect).

Example 5: CD spectra of individual peptides or peptide-modified nanoparticles at different pH conditions (display of pH responsiveness and necessity for matrix of membrane structure)

The peptide obtained in productive example 1 alone and the liposome 1 obtained in example 1 (each having a peptide concentration of 20 μ M) were suspended in PBS (-) having different pH, and CD (circular dichroism) spectra were recorded on a J-720WI spectropolarimeter (manufactured by JASCO corporation). Subsequently, the composition of the secondary structure in the broad spectrum was predicted using analytical software (see JWSSE-480; Molecular Membrane Biology, July, August2007;24(4): 282-. Fig. 9 shows the results.

In fig. 9 below, "α helix" denotes an α helix structure; "curls" means random curls (no clear secondary structure is formed); and "corners" indicate curved structures.

According to the results obtained using the peptide alone, the CD spectra obtained at pH7.4 and 6.5 were almost the same, and the spectrum at pH 6.0 was greatly changed. This confirms that when the peptide is used alone, structural change does not occur unless the pH is lowered to 6.0.

Considering the CD spectrum of liposome 1, the spectrum of liposome 1 at pH7.4 is different from that obtained using the peptide alone. It was thus revealed that the state of the secondary structure is different from that when the peptide was used alone, since the peptide is located on the lipid membrane. Furthermore, the change in pH from 7.4 to 6.5 caused a large change in CD spectrum, with almost the same results as obtained at pH 6.0 and 5.5. This clarifies the fact that the peptide located on liposome 1 undergoes large structural changes due to small pH changes. Thus confirming that particles or membrane structures such as liposomes or micelles are necessary to ensure sensitivity of the peptide to small pH changes.

Example 6: evaluation results of pH responsiveness with respect to peptides having scrambled sequences and with respect to peptides of different lengths (particle size, zeta potential, cellular uptake, etc.)

(1) As described in the preparation method of example 1, stearylated peptides of production examples 2 to 4 were added to respective liposome suspensions containing EPC and DOTAP each at a ratio of 8:1 (molar ratio), and the mixture was incubated, thereby preparing peptide-modified liposomes 2 to 4, the surfaces of each of which were modified with the respective peptides.

(2) The particle size (size) and surface potential (zeta potential) of each peptide-modified liposome diluted and suspended in buffer solutions of different pH were measured by a Zetasizer Nano potential analyzer manufactured by Malvern Instruments ltd.

Table 2 shows the results of peptide-modified liposome 4 obtained using the peptide of production example 4.

TABLE 2

Liposome 4, which does not have a unit repeat that starts with His and ends with an acidic amino acid, has a positive surface potential at all phs. Thus, liposomes 4 are completely different from the liposomes of the present invention in their properties.

Cellular uptake of liposome 4 was assessed by FACS. At pH7.4, a large amount of liposome 4 is taken up by the cells; even when the pH was lowered to 6.5, almost the same amount was taken up by the cells (fig. 10). These results are consistent with the results obtained for the above-mentioned surface potential, indicating that liposome 4, which does not have a repeat of units starting with His and terminating with an acidic amino acid, does not respond to small pH changes, even with the same constituent amino acids.

(3) Liposomes 2a, 2b and 2c were prepared as described in the method of example 1 using the peptide of production example 2 having 4 residues shorter than the peptide obtained in production example 1 in amounts of 5, 6 and 7mol% of the lipid content for modification, respectively, and the surface potential was measured. As a result, all liposomes showed similar tendencies as liposome 1 (table 3).

TABLE 3

Peptide (4 residue shortened) modified liposomes

(peptide modification: 5mol% lipid content)

Peptide (4 residue shortened) modified liposomes

(peptide modification: 6mol% lipid content)

In addition, cellular uptake was assessed by FACS. As a result, when the pH was 7.4, the amount of cellular uptake of all of these liposomes was almost the same as in the case where untreated cells were involved, i.e., almost no liposome was taken up by the cells; however, when the pH is 6.5, the amount of cellular uptake increases.

This demonstrates that even when liposomes are modified with peptides shortened by 4 residues, they respond to small pH changes, thereby significantly increasing their affinity for cells.

(4) In addition, as described in the method of example 1, using the peptide of production example 3 having 8 residues shorter than the peptide of production example 1, liposomes 3a, 3b and 3c were prepared in amounts of 5, 7.5 and 10mol% of the lipid content for modification, respectively, and the surface potential of each liposome was measured (table 4).

TABLE 4

Peptide (8 residue shortened) modified liposomes

(peptide modification: 5mol% lipid content)

Peptide (8 residue shortened) modified liposomes

(peptide modification: 7.5mol% lipid content)

Peptide (8 residue shortened) modified liposomes

(peptide modification: 10mol% lipid content)

As a result, all of these liposomes showed large differences in surface potential between pH values of 7.4 and 6.5. Specifically, the liposome 3c in which 10mol% of the lipid content is modified has a large surface potential.

Claims (12)

1. Nanoparticles comprising a peptide and a particle-forming component, wherein

The particle-forming component forms a liposome or a micelle,

wherein the peptide comprises 2 to 8 units represented by the following formula (I):

His-(AA₁)(AA₂)(AA₃)-Glu/Asp (I)，

wherein His is histidine; Glu/Asp is glutamic acid or aspartic acid; and AA₁、AA₂And AA₃Are the same or different and each represents Gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, or Asn, and

wherein the amino acid sequence of each unit is the same or different.

2. The nanoparticle of claim 1, wherein the amino acid between the His and the acidic amino acid is any amino acid selected from the group consisting of: gly, Ala, His, Cys, and Ser.

3. The nanoparticle of claim 1, wherein the peptide has the sequence of any one of SEQ ID Nos 1 to 3.

4. The nanoparticle of any one of claims 1 to 3, wherein the peptide has a hydrophobic group at a terminus so as to be retained by the liposome or micelle.

5. The nanoparticle of claim 4, wherein the hydrophobic group is C_12-24Hydrocarbyl or C_12-24An acyl group.

6. The nanoparticle of any one of claims 1 to 5, wherein the particle-forming component comprises a phospholipid.

7. The nanoparticle of any one of claims 1 to 6, wherein the particle-forming component forms a liposome.

8. A substance introducing agent comprising the nanoparticle of any one of claims 1 to 7.

9. A peptide compound represented by the following formula (II):

R¹-(Z¹)_l-[His-(AA₁)(AA₂)(AA₃)-Glu/Asp]_n-(Z²)_m-R²(II)，

wherein His is histidine; Glu/Asp is glutamic acid or aspartic acid; AA₁、AA₂And AA₃Are identical or different and each represents Gly, Ala, His, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln or Asn; n represents an integer of 2 to 8; l and m are the same or different and each represents 0 or 1; r¹Is C_12-24Hydrocarbyl or C_12-24An acyl group; r²Is OH or a C-terminal protecting group; and Z is¹Or Z²Represents a linker arm consisting of 1 to 8 amino acids selected from the following amino acids: gly, Ala, Leu, Ile, Val, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Gln, and Asn,

the peptide compounds contain a total of 10 to 60 amino acids.

10. The peptide compound of claim 9, wherein the amino acid between His and an acidic amino acid is any amino acid selected from Gly, Ala, His, Cys and Ser.

11. The peptide compound according to claim 10, wherein the peptide in the peptide compound represented by the formula (II) has a sequence of any one of SEQ id nos 1 to 3.

12. The peptide compound of claim 10 or 11, wherein R¹Is C_12-24An acyl group.