Antiviral nanoparticles and their use against influenza viruses

Abstract

The present invention relates to antiviral nanoparticles comprising a trisaccharide moiety and the use thereof against influenza viruses.

Description

Technology Field

[0001] The present invention relates to antiviral nanoparticles comprising a trisaccharide moiety and the use thereof against influenza viruses. Background Technology

[0002] Influenza is one of the most contagious viruses. Every year, various influenza strains infect large portions of animal and human populations, putting infants, the elderly, and immunocompromised individuals at risk of hospitalization and death due to influenza-related complications. Consequently, seasonal influenza has a significant socioeconomic impact. Indeed, respiratory diseases can account for a substantial portion of total healthcare costs in both developed and major developing countries. Because influenza mutates very rapidly, vaccine development remains a significant challenge. Vaccine development poses even greater challenges when focusing on infrequent epidemics rather than annual ones. In such cases, the development time for a new vaccine—averaging six months—constitutes a serious risk. Furthermore, even if a vaccine exists, achieving reasonable vaccination coverage is far from a certainty. Therefore, the risk of new epidemics, such as the Spanish flu, remains present and is recognized as one of the greatest threats to global health.

[0003] Naturally, the second line of defense following vaccines is antiviral drugs. Many anti-influenza drugs are currently approved: neuraminidase inhibitors such as zanamivir and oseltamivir, ion channel inhibitors such as amantadine, fusion inhibitors such as umifenovir (only in Russia and China), and polymerase inhibitors such as baloxavir marboxil, recently approved in the U.S. and Japan. However, it is recognized that the efficacy of current drugs falls far short of expectations. Concerns regarding these drugs range from serious side effects to the emergence of drug-resistant viruses after short-term use. Given the importance of this issue, many other drugs are currently undergoing clinical trials. Most of these drugs are monoclonal antibodies that inhibit the virus from fusing into host cells. While promising, their manufacturing processes are likely to incur significant costs.

[0004] There are quite a few research lines regarding the development of molecules that target conserved regions of viruses. Additionally, finding antiviral (i.e., irreversible) drugs with limited toxicity is very difficult. Recently, peptides isolated from frog skin were discovered to possess these characteristics, but their EC 50 It is only in micromoles, and their toxicity is debatable.

[0005] The interaction between viral hemagglutinin (HA) and sialic acid (SA)-containing glycoproteins in host cells is a key step in influenza infection. The binding affinity between SA and HA is low and is compensated for by multivalent binding. Inspired by this natural phenomenon, there have been several attempts to inhibit the influenza virus using SA-coated multivalent materials such as polymers, dendrimers, and nanoparticles.

[0006] Reuter et al. synthesized sialic acid-decorated polymerizable materials of various structures, such as linear polymers, comb-branched polymers, and dendrons, and tested them against different strains of influenza A virus. It was found that only high molecular weight (>100 kDa) branched structures inhibited one strain of influenza virus, X-31, at micromolar concentrations of sialic acid. Papp et al. tested the inhibitory activity of sialic acid-functionalized gold nanoparticles (NPs) against the influenza virus X-31 strain and reported that NPs with a core diameter of 14 nm exhibited superior inhibitory activity compared to NPs with a core diameter of 2 nm. However, in this study, they did not mention the NP concentration that inhibits the virus. The same group also synthesized sialic acid-decorated glycostructures of different sizes and inhibited the X-31 strain at millimolar concentrations.

[0007] Human influenza viruses are known to preferentially bind to α2,6-linked SA on glycoproteins. More recently, it has been demonstrated that polyvalent substances containing α2,6-sialyllactose inhibit influenza viruses at low micromolar concentrations. Tang et al. synthesized a brush polymer containing α2,6-sialyllactose that inhibited the influenza A PR8 strain with sialic acid units at micromolar concentrations. Kwon et al. decorated PAMAM dendrimers with α2,6-sialyllactose and inhibited several influenza strains such as influenza A PR8, CAL 09, and NWS 33.

[0008] The micromolar concentration of a substance that inhibits the influenza virus in vitro is still too high, and the in vivo concentration would be much higher. Furthermore, most entry inhibitors are antiviral; the virus becomes infectious again when the virus-substance complex is diluted in vitro.

[0009] In the absence of a broad-spectrum, effective vaccine, there is an unmet need for drugs against influenza. An ideal anti-influenza drug must have a broad spectrum, target highly conserved portions of the virus, and possess an irreversible effect—that is, it must be virucidal at low concentrations (to prevent loss of efficacy due to fluid dilution)—and must be clearly non-toxic.

[0010] To solve this problem, the present invention provides nanoparticles that strongly interact with HA, irreversibly inhibit the infectivity of the influenza virus at low concentrations, and exhibit very low toxicity.

[0011] One embodiment of the present invention provides a virucidal nanoparticle comprising a core and a plurality of ligands covalently connected to the core, wherein at least a portion of the ligands comprises a trisaccharide moiety, and wherein:

[0012] - The above core is cyclodextrin, and

[0013] - The above ligand is an identical or different optionally substituted alkyl ligand, and

[0014] - Each trisaccharide moiety is selected from 3-sialyl-N-acetyllactosamine (3'SLN) and 6-sialyl-N-acetyllactosamine (6'SLN).

[0015] Another embodiment of the present invention provides a virucidal nanoparticle represented by formula (I):

[0016]

[0017] Here

[0018] m is 2~8, and

[0019] n is 2 to 28 or 4 to 13.

[0020] Another embodiment of the present invention provides a virucidal nanoparticle represented by formula (II) or a pharmaceutically acceptable salt thereof:

[0021]

[0022] Here:

[0023] Each R is independently, optionally substituted alkyl ligand, wherein at least two of the ligands have a trisaccharide moiety selected from the group comprising 3-sialyl-N-acetyllactosamine (3'SLN) and 6-sialyl-N-acetyllactosamine (6'SLN), or at least one of the ligands has 3'SLN and the other has 6'SLN;

[0024] Each R' is, independently, H, -(CH2) y -COOH, -(CH2) y -SO3 - , polymer or water-soluble moiety;

[0025] x is 6, 7, or 8; and

[0026] y is an integer from 4 to 20.

[0027] Further embodiments of the present invention provide a pharmaceutical composition comprising an effective amount of one or more of the antiviral nanoparticles of the present invention and at least one pharmaceutically acceptable excipient, carrier, and / or diluent.

[0028] Further embodiments of the present invention provide the antiviral nanoparticles of the present invention for use in treating and / or preventing influenza virus infections and / or diseases associated with influenza viruses.

[0029] Further embodiments of the present invention provide a viscous composition comprising an effective amount of one or more viscous nanoparticles of the present invention and optionally at least one suitable aerosol carrier.

[0030] Further embodiments of the present invention provide a disinfection and / or sterilization method comprising using the antiviral composition of the present invention or the antiviral nanoparticles of the present invention.

[0031] Further embodiments of the present invention provide an apparatus comprising a viscous composition of the present invention, or one or more viscous nanoparticles of the present invention, and means for applying or dispensing said viscous composition or viscous nanoparticles.

[0032] Further embodiments of the present invention provide the use of the viscous nanoparticles of the present invention or the viscous composition of the present invention for sterilization and / or disinfection. Brief explanation of the drawing

[0033] Fig. 1 Silver shows the chemical structures of NPs with different ligand compositions. Average NP diameter: 2.9±0.9 nm. Fig. 2 ) shows TEM images of the H3N2 virus Vic / 11 strain: virus after 1 hour incubation with no NPs (A), LD 6'SLN NPs (B), and PEG(5) NPs (C). Fig. 3 Chemical structure of C15-6'SLN modified β-CD (a), EC of modified CD against different influenza strains 50 Shows concentration (b), virucidal activity against Neth / 09 strain (c), in vitro experiment in MucilAir (d), and illustration of a modified CD interacting with a HA spherical head (e). Fig. 4 shows an exemplary modified cyclodextrin. The number of 6'SLN or 3'SLN per β-CD is 1 It is the average number calculated by H NMR. The representative chemical structure of the modified cyclodextrin was constructed based on the NMR results. EC 50 represents the half-inhibitory concentration of MDCK cells against A / Netherlands / 2009 (H1N1) at 24 hpi (Fig. 7). N / A: Not evaluated. To facilitate reference in these 3D structures, some of the backward cyclodextrin sugars, ligands, and trisaccharides are not shown. Fig. 5 to characterize the modified cyclodextrin shown in FIG. 4 1 This shows that H NMR studies were performed. The average number of 6'SLN or 3'SLN per β-cyclodextrin was calculated by comparing the unique peak (◆) of trisaccharide with the unique peak (●) of β-cyclodextrin. Both peaks correspond to a single hydrogen. Fig. 6 This shows dose-response curves demonstrating the antiviral activity of C6-6' (A), C14-6' (B), C11-3' (C), and C1-6' (D) against A / Netherlands / 2009 H1N1. The above results are the average of two independent experiments performed in duplicate. Fig. 7 This shows dose-response curves demonstrating the antiviral activity of C11-6' against B / Wisconsin / 2010 (A), A / Clinical / 2018 H1N1 (B), A / Singapore / 2004 H3N2 (C), and B / Clinical / 2018 (D). The above results are the average of two independent experiments performed in duplicate. Fig. 8 This shows the dose-response curve (A) demonstrating the antiviral activity of C11-3' against avian strain A / turkey / Turkey / 2005 H5N1 (along with the C11-6' control) and the dose-response curve (B) demonstrating the antiviral activity of C11-3' against avian strain A / Turkey / Italy / 977 / 1999 H7N1. For Graph A, infection was quantified using FACS and ICC methods. The above results are the average of two independent experiments performed in duplicate. Fig. 9 This shows a comparison of the in vitro antiviral activities of C11-6' and P8-6'. Panel ( a ) and ( bThe graph on the left shows the inhibitory activity of each compound against A / NL / 09, overlaid with the results of the cell viability analysis. All compounds exhibit very similar behavior. The graph on the right of these panels shows the results of the virucidal (i.e., dilution) test. On the axis of the figure ffu represents a focus forming unit NT Note that indicates unprocessed. c )at C11-6' It was tested against the following virus strains: A / Singapore / 2004 (H3N2), B / Wisconsin / 2010, and A / Clinical / 2018 (H1N1). The above results are the mean and SEM of two independent experiments performed in duplicate. Fig. 10 [It is for A / Netherhands / 2009 (H1N1)] C14-6' (A), C6-6' (B) and C11-3' (C) shows the antiviral activity. The experiment was performed at a compound concentration of 100 μg / ml. The above results are the average of two independent experiments and SEM. Fig. 11 silver C11-6' and P8-6' It shows a comparison of the in vitro inhibitory activity of. C11-6' While provided complete protection against clinically epidemic H1N1 O9 strains under co-treatment conditions, P8-6' provided only slight protection in the early stages of infection ( a ). In Fig. 11(b), the immunofluorescence at 7 days post-infection (co-treatment condition) is C11-6' Confirms the protection provided by. Red: Monoclonal antibody influenza A, Blue: DAPI, Green: β-IV-tubulin (marker of ciliated cells). The thickness of each tissue is indicated at the bottom of the corresponding image ( b ). C11-6' It showed high efficacy even under post-treatment conditions ( cThe results in (a) and (c) are the average and SEM of 2 to 4 independent experiments performed in duplicate. The image in (b) is a representative of 10 images taken for each condition. Fig. 12 It shows LDH release from infected tissue. At infection C11-6' (50 µg) or P8-6' Tissues were infected and treated with [the appropriate method]. LDH measurements were performed on apical washes conducted at 96 and 24 hpi. The above results are from two independent experiments on H1N1 and H1N1 C11-6' and P8-6' This is the average and SEM of a single experiment performed redundantly on. Fig. 13 This shows a long-term co-treatment experiment. Tissues were infected and treated with C11-6' (50 µg) upon infection. To evaluate daily viral load, daily peak washes were collected from the previous day's wash during the first 5 days, and then on days 9, 17, and 23. The results above are the average of a single experiment performed with overlap. Fig. 14 It exhibits in vitro toxicity. The tissues are of different doses. C11-6' Alternatively, the tissues were treated with the daily addition of an equal volume of medium or Triton 5%. 96 hours after treatment, A) MTT assay, B) LDH assay to assess tissue viability, C) assessment of trans-epithelial resistance, and D) ELISA assay to assess the release of pro-inflammatory cytokines were performed on the tissues. LDH and ELISA were performed on the collected basal medium. The results are the mean of two independent experiments and SEM. Fig. 15 Is In vivo antiviral activity of C11-6' It shows. a to c Mice were intranasally treated with PBS or C11-6' at the time of infection with A / NL / 09 or 48 hours later. Viral load was quantified at 48 and 96 hours post-infection (a), and morbidity (loss of temperature (b) and body weight (c)) in infected mice was monitored daily. d to f Mice were intranasally treated with PBS or C11-6' daily (14 µg / mouse) 6 hours post-infection for 3 days. The morbidity (de) and survival rate (f) of infected mice were monitored daily. The results are expressed as mean ± SEM. Arrows indicate treatment time. Fig. 16 β-cyclodextrin, 6'SLN-β-ethylamine and C11-6' of 1 It shows the stacked H NMR spectrum. Fig. 17 It shows the DOSY spectrum of C11-6', which proves that there are no unbonded trisaccharides in the resulting compound. Fig. 18 It shows the gating strategy performed for FACS. Specific details for implementing the invention

[0034] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for the purpose of disclosing them prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment that the present invention is not qualified to precede such publications due to prior art. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0035] In the event of a conflict, this specification, including definitions, will control it.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the subject matter of this specification belongs. As used herein, the following definitions are provided to facilitate understanding of the invention.

[0037] The term "include" is generally used in the sense of inclusion, that is, to allow the presence of one or more features or components. Additionally, as used in the specification and claims, the language "includes" may include similar embodiments described in terms of being "composed" and / or "essentially composed."

[0038] As used in the specification and claims, the term "and / or" used in phrases such as "A and / or B" in this specification is intended to include "A and B", "A or B", "A", and "B".

[0039] As used in the specification and claims, the singular forms "one (a)," "one (an)," and "the" include references to the plural form unless the context clearly indicates otherwise.

[0040] As used in the specification and claims, the term "at least one" used in phrases such as "at least one C atom" may mean "one C atom," "two C atoms," or more C atoms.

[0041] As used herein, the term "virtatic" refers to a characteristic of antiviral efficacy measured by in vitro tests demonstrating the reversible inhibition of viral infectivity following interaction with an antiviral composition. The interaction inhibits infectivity, for example, by binding to the virus or otherwise interfering with the viral surface ligands. However, once the interaction is terminated (e.g. by dilution), the virus may regain infectivity in the absence of any added substance or condition that promotes viral reconstitution.

[0042] As used herein, the term “salviral” refers to a characteristic of antiviral efficacy measured by in vitro tests demonstrating the irreversible inhibition of viral infectivity following interaction with an antiviral compound or composition. The interaction inhibits infectivity, for example, by binding to the virus or otherwise interfering with the viral surface ligands. However, in the absence of any added substance or condition that promotes viral reconstitution even after the interaction has ended (e.g., by dilution), it is essentially impossible for the virus to regain infectivity.

[0043] As used herein, the term “biocompatibility” refers to compatibility with living cells, tissues, organs, or systems, and the absence of a significant risk of injury, toxicity, or rejection by the immune system.

[0044] As used herein, "nano" as used in "nanoparticle" refers to nanometer size, such as particles having a nanometer size, and is not intended to convey any specific shape limitations. In particular, "nanoparticle" includes nanospheres, nanotubes, nanoboxes, nanoclusters, nanorods, etc. In certain embodiments, the nanoparticles and / or nanoparticle cores considered herein generally have polyhedral or spherical geometry.

[0045] As used herein, “influenza” refers to sialic acid-seeding, airborne (human or animal) RNA viruses, such as influenza A virus, influenza B virus, influenza C virus, and influenza D virus. Influenza A virus includes the following serotypes: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and H6N1.

[0046] As used herein, the term "alkyl" refers to a straight-chain hydrocarbon containing 1 to 50 carbon atoms, preferably 4 to 30 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.

[0047] As used in this specification, the term “carboxyalkyl” refers to a carboxyl group added to a parent molecular moiety through an alkyl group as defined in this specification.

[0048] Although α2,6-linked sialic acid is known to interact with human influenza viruses, the exact glycan sequences that provide high-affinity binding have remained unclear in the literature. The inventors have demonstrated that most human influenza viruses bind with high affinity to glycans terminated by two or more, preferably three or four, trisaccharide moiety, particularly 6-sialyl-N-acetylactosamine (6'SLN) or 3-sialyl-N-acetylactosamine (3'SLN).

[0049] One embodiment of the present invention provides a viscous nanoparticle comprising a core and a plurality of ligands covalently connected to the core, wherein at least a portion of the ligands comprises a trisaccharide moiety, and wherein

[0050] - The core is a metal nanoparticle or an organic material, wherein preferably the metal nanoparticle is selected from the group comprising gold nanoparticles, iron oxide nanoparticles, silver nanoparticles, platinum nanoparticles, cobalt nanoparticles, zinc nanoparticles, silica nanoparticles, cadmium selenide nanoparticles, gold-silver alloy nanoparticles, aluminum oxide nanoparticles, copper oxide nanoparticles, magnesium oxide nanoparticles, nickel oxide nanoparticles, titanium dioxide nanoparticles, and zinc oxide nanoparticles, more preferably the metal nanoparticle is a gold nanoparticle, wherein the organic material is selected from the group comprising cyclodextrin, polymers, dendrimers, and dendrons, and preferably the organic material is a cyclodextrin.

[0051] - The ligand is an identical or different optionally substituted alkyl ligand or polyethylene glycol (PEG) ligand. Preferably, the optionally substituted alkyl ligand is an optionally substituted C4-C 30 It is an alkyl ligand, and more preferably, the optionally substituted alkyl ligand is an optionally substituted C4-C 30 It is a carboxyalkyl; preferably, the PEG-based ligand is selected from the group comprising PEG3, PEG4, PEG5, PEG6, PEG7, and PEG8; more preferably, the ligand is derived from the group comprising polyethylene glycol 5 (PEG5), 16-mercaptohexadeconic acid (C15), and 11-mercaptoundecanic acid, and

[0052] - The trisaccharide moiety is 3-sialyl-N-acetyllactosamine (3'SLN) and / or 6-sialyl-N-acetyllactosamine (6'SLN).

[0053] In the context of the present invention, a trisaccharide moiety is exposed on a ligand covalently bonded to the outer surface of a nanoparticle (NP) in such a way that any other ligand does not interfere with the interaction between the trisaccharide moiety and the influenza virus.

[0054] The average diameter of the core is in the range of about 1.0 nm to about 200 nm, preferably 1 nm to 5 nm, and most preferably 1.5 nm to 3 nm. The total nanoparticle size has an average particle diameter of 3 nm to 250 nm, or 3 nm to 200 nm, preferably 3 nm to 10 nm, and more preferably 4.5 nm to 6 nm.

[0055] In some embodiments, the core within the antiviral nanoparticle of the present invention is an organic material, preferably a polymer, wherein the polymer is selected from the group comprising polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polylactic acid (PLA), polyglycolide (PGA), polydioxanone (PDO), and poly(lact-co-glycolic acid).

[0056] In some embodiments, the core within the antiviral nanoparticle of the present invention is a dendrimer selected from the group comprising organic materials, preferably poly(amidoamine) (PAMAM) and bis-MPA.

[0057] In some embodiments, the core within the antiviral nanoparticle of the present invention is a dendron selected from the group comprising organic material, preferably poly(amidoamine) (PAMAM) and bis-MPA.

[0058] In a preferred embodiment, the core of the antiviral nanoparticle of the present invention is an organic material that is cyclodextrin.

[0059] Cyclodextrin (CD) is a naturally occurring cyclic glucose derivative composed of alpha (14)-linked glucopyranoside units. Their cyclic structure creates a truncated cone shape in which the first hydroxyl group of the glucose unit is on the narrow face and the second hydroxyl group is on the wide face. Each face can be easily functionalized independently. The most commonly used natural CDs have 6, 7, and 8 glucopyranoside units and are referred to as alpha, beta, and gamma cyclodextrin, respectively. The preferred cyclodextrin is beta. Due to the cyclic structure of CD, it has cavities capable of forming supramolecular inclusion complexes with guest molecules. Because CDs occur naturally, are easily functionalized, have cavities for guest inclusion, and are biocompatible, their use has been found in many commercial applications, including drug delivery and air fresheners. Different reactivity of each face of CD has been utilized for the synthesis of a wide range of modified cyclodextrins. The first plane of the CD is modified more easily because the degree and location of substitution can be controlled. CD derivatives containing excellent leaving groups, such as halogenated CDs, are important intermediates in CD functionalization. By replacing all first hydroxyl units of the CD with iodo units, an intermediate is provided that enables complete functionalization of the first plane, while the second hydroxyl and the rigid truncated cone shape remain intact. In one embodiment, heptakis-6-iodo-6-deoxy-beta-cyclodextrin was synthesized and then reacted with mercaptoundecaosulfonate (MUS) to produce a functionalized CD having undecanaosulfonate groups on the first plane. Subsequently, the second plane of the cyclodextrin can be independently modified to introduce additional solubilizing groups, dye molecules, polymers, etc. In addition, the size of β-CD (d~1.5 nm) falls within the preferred nano-size for the core of the present invention and matches well with the HA spherical head (~5 nm).Beta-cyclodextrin has a hard chemical structure that is believed to contribute to antiviral activity and can have up to 7 trisaccharide-containing ligands along the narrow side, preferably 3 to 4 trisaccharide-containing ligands, where 3 is the number of sialic acid binding sites in the spherical head of the influenza virus HA.

[0060] The antiviral nanoparticles of the present invention may also be purified single molecules or compounds intended to be included within the scope of the present invention.

[0061] One embodiment of the present invention provides a virucidal nanoparticle comprising a core and a plurality of ligands covalently connected to the core, wherein at least a portion of the ligands comprises a trisaccharide moiety, and wherein

[0062] - The core is preferably a cyclodextrin selected from the group comprising alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, or a combination thereof, and

[0063] - The above ligand is an identical or different optionally substituted alkyl ligand, preferably an optionally substituted C4-C 30 Alkyl ligands, more preferably optionally substituted C6-C 15 It is an alkyl ligand, and

[0064] - Each trisaccharide moiety is selected from 3-sialyl-N-acetyllactosamine (3'SLN) and 6-sialyl-N-acetyllactosamine (6'SLN).

[0065] In some embodiments of the antiviral nanoparticles of the present invention, some or all of the ligand comprises a 3'SLN, some or all of the ligand comprises a 6'SLN, and some but not all of the ligand does not comprise a trisaccharide moiety.

[0066] The antiviral nanoparticles of the present invention (wherein the core is cyclodextrin) can be represented by formula (I):

[0067]

[0068] Here

[0069] m is 2 to 8, preferably m is 2 to 7 or 2 to 6, more preferably m is 3 or 4, and

[0070] n is 2 to 28 or 4 to 13 or 4 to 30 or 6 to 15, preferably 2 to 28 or 4 to 13, and in some embodiments, n is 2 or 4 or 6 to 13 or 28 or 30, and

[0071] Preferably, the cyclodextrin is selected from the group comprising alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, or a combination thereof.

[0072] Another embodiment of the present invention provides a virucidal nanoparticle represented by formula (II) or a pharmaceutically acceptable salt thereof:

[0073]

[0074] Here

[0075] Each R is independently, optionally substituted alkyl ligand, wherein at least two of the ligands have a trisaccharide moiety selected from the group comprising 3-sialyl-N-acetyllactosamine (3'SLN) and 6-sialyl-N-acetyllactosamine (6'SLN), or at least one of the ligands has 3'SLN and the other has 6'SLN; preferably, the optionally substituted alkyl ligand is an optionally substituted C4-C 30 Alkyl ligands, more preferably optionally substituted C6-C 15 Optionally substituted C6-C containing alkyl ligands or 6'SLNs 15 It is an alkyl ligand;

[0076] Each R' is, independently, H, -(CH2) y -COOH, -(CH2) y -SO3 - , polymer or water-soluble moiety; preferably R' is H; preferably R' is, independently, H, -(CH2) y -COOH, -(CH2) y -SO3 - , or is a polymer; preferably R' is, independently, -(CH2) y -COOH, -(CH2) y -SO3 - , or is a polymer; preferably R' is, independently, H, -(CH2) y -COOH, or -(CH2) y -SO3 - and; preferably R' is, independently, -(CH2) y -COOH, or -(CH2) y -SO3 - And;

[0077] x is 6, 7, or 8; and

[0078] y is an integer of at least 4 to about 20, preferably y is at least 4, preferably y is 4 to 20, preferably y is 7 to 11, and most preferably y is 10. In another embodiment, y is at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11. In another embodiment, y is at most 100, at most 70, at most 50, at most 25, at most 20, or at most 15.

[0079] The polymer in the antiviral nanoparticles of the present invention may be selected from both synthetic and natural polymers. In one embodiment of the present invention, the synthetic polymer is selected from the group comprising, but not limited to, poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(n-butyl acrylate), poly-(α-ester), (PEG-b-PPO-b-PEG), poly(N-isopropylacrylamide) (pNIPAAM), polylactglycolic acid (PLGA), and / or combinations thereof. In another embodiment of the present invention, the natural polymer is selected from the group comprising dextran, dextrin, glucose, cellulose, and / or combinations thereof.

[0080] In some embodiments of the antiviral nanoparticles of the present invention, the trisaccharide moiety is preferably a 6'SLN specific to human influenza strains. In other embodiments of the antiviral nanoparticles of the present invention, the trisaccharide moiety is preferably a 3'SLN specific to avian influenza strains.

[0081] The ligand (or ligand compound) of the antiviral nanoparticles of the present invention is typically sufficiently long (at least 2, or at least 4, or at least 6 carbon atoms) and hydrophobic.

[0082] Typically, in the context of the present invention, the optionally substituted alkyl ligand is selected from the group comprising hexane-, pentane-, octane-, undecane-, and hexadecane-based ligands.

[0083] Substituted alkyl ligand of the antiviral nanoparticles of the present invention, substituted C4-C 30 Alkyl ligands, and substituted C4-C 30Carboxyalkyl includes alkenyl, alkenylthio, alkenyloxy, alkoxy, alkoxyalkoxy, alkoxyalkoxyalkoxy, alkoxyalkoxyalkyl, alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkoxy, alkoxycarbonylalkyl, alkoxysulfonyl, alkyl, alkylamidoalkyl, alkylcarbonyl, alkylcarbonylalkoxy, alkylcarbonylalkylthio, alkylcarbonyloxy, alkylcarbonylthio, alkylsulfinyl, alkylsulfinylalkyl, alkylsulfonyl, alkylsulfonylalkyl, alkylthio, alkylthioalkyl, alkylthioalkoxy, alkyninyl, alkyninoxy, alkyninthio, aryl, arylcarbonyl, aryloxy, arylsulfonyl, carboxy, carboxyalkoxy, carboxyalkyl, cyano, It is substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group comprising cyanoalkoxy, cyanoalkyl, cyanoalkylthio, 1,3-dioxoranyl, dioxanyl, dithianyl, ethylenedioxy, formyl, formylalkoxy, formylalkyl, haloalkenyl, haloalkenyloxy, haloalkoxy, haloalkyl, haloalkynyl, haloalkynyloxy, halogen, heterocycle, heterocyclocarbonyl, heterocyclooxy, heterocyclosulfonyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, mercapto, mercaptoalkoxy, mercaptoalkyl, methylenedioxy, and nitro. Preferably, the substituted alkyl ligand, the substituted C4-C 30 Alkyl ligands, and substituted C4-C 30 The carboxyalkyl group is substituted with a mercapto group (replacing the corresponding oxygen of the unmodified cyclodextrin). The preferred substituted alkyl ligand is a C4-C 30 or C2 to C 28 or C4 to C 13 It is an alkylamidoalkyl substituted with an alkyl ligand.

[0084] The percentage ratio between the ligand and the ligand containing the trisaccharide moiety is 75%:25% to 95%:5%; preferably 88%:12%.

[0085] In the context of this disclosure, “a plurality of ligands” refers to a viviparous nanoparticle core partially or completely coated by a plurality of ligands of the present invention, wherein at least a portion of said ligands comprises the trisaccharide moiety of the present invention. The coating may be homogeneous, unstructured, or structured. In some embodiments, the viviparous nanoparticles comprise ligands comprising the trisaccharide moiety of the present invention in a very high density (HD), for example, 25% of the total ligands. In some embodiments, the viviparous nanoparticles are nm 2 Each comprises about 2 to about 5 ligands of the present invention, wherein at least some of the ligands comprise a trisaccharide moiety. In another embodiment, the antiviral nanoparticles are nm 2 The present invention comprises four ligands, wherein at least some of these ligands comprise a trisaccharide moiety.

[0086] In some embodiments of the present invention, a plurality of ligands of the present invention comprises a mixture of at least two structurally different ligands, such as polyethylene glycol 5 (PEG(5)) and 16-mercaptohexadeconic acid (C15). As used herein, the term “mixture of at least two structurally different ligands” refers to a combination of two or more ligands of the present invention as defined above, wherein the ligands differ from each other in their chemical composition at at least one position. The mixture can advantageously be organized such that a ligand not containing a trisaccharide moiety provides an optimal spacing for a ligand containing a trisaccharide moiety and does not interfere with the interaction between the trisaccharide moiety and the influenza virus HA. Thus, there will be a percentage ratio between the ligand not containing a trisaccharide moiety and the ligand containing a trisaccharide moiety, in the range of about 75:25 to about 95:5, preferably about 88:12.

[0087] According to one embodiment of the present invention, gold nanoparticles (NPs) with a ligand mixed with PEG (5) and 16-mercaptohexadeconic acid (C15) were synthesized. PEG (5) improves the water solubility of the NPs, while C15 is a ligand for grafting trisaccharides. The selection of the ligands was based on two reasons: 1) C15 is long enough to target three sialic acid binding sites located ~4 nm apart on the HA, and 2) carbon-based hard ligands improve the activity of the antiviral.

[0088] The ligand is generally present on the surface of the core in an amount that optimizes the binding of the trisaccharide moiety to influenza hemagglutinin. The core is typically nm 2 It has 4 ligands per gram. In some embodiments, the core is nm 2 Each has about 2 to about 5 ligands.

[0089] A significant advantage of the antiviral nanoparticles of the present invention is that detailed toxicity analysis results showed no changes in tissue structure or release of pro-inflammatory cytokines. In vivo tests demonstrated that treatment with the antiviral nanoparticles of the present invention significantly improved the health status of infected mice and significantly reduced the viral load in the lungs, regardless of the addition of drugs before or after infection.

[0090] Another embodiment of the present invention discloses a pharmaceutical composition comprising an effective amount of one or more of the antiviral nanoparticles of the present invention and at least one pharmaceutically acceptable excipient, carrier, and / or diluent.

[0091] Regarding appropriate excipients, carriers, and diluents, refer to standard literature describing them, for example, "Comprehensive Medicinal Chemistry," chapter 25.2 of Vol. 5, Pergamon Press, 1990, and "Lexikon der Hilfsstoffe f" by HP Fiedler. Refer to "r Pharmazie, Kosmetik und angrenzende Gebiete", Editio Cantor, 2002. The term "pharmaceuticalally acceptable carriers, excipients and / or diluents" means carriers, excipients, or diluents useful for producing pharmaceutical compositions that are generally safe and have acceptable toxicity. Acceptable carriers, excipients, or diluents include those that may be accepted for human pharmaceutical uses as well as veterinary uses. "Pharmaceutically acceptable carriers, excipients, and / or diluents" as used in the specification and claims comprises both of one or more of such carriers, excipients, and / or diluents.

[0092] Optionally, the pharmaceutical composition of the present invention further comprises one or more additional active agents, preferably antiviral agents.

[0093] The antiviral nanoparticles of the present invention used in the method of the present invention may be incorporated into various formulations and pharmaceuticals for therapeutic administration. More specifically, antiviral nanoparticles as provided herein may be formulated into pharmaceutical compositions in combination with suitable, pharmaceutically acceptable carriers, excipients, and / or diluents, and may be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, pills, powders, granules, drases, gels, slurries, ointments, solutions, suppositories, injections, inhalants, and aerosols. Thus, administration of antiviral nanoparticles may be achieved in various ways, including oral, buccal, inhalation (lung, nose), rectal, parenteral, intraperitoneal, intradermal, transdermal, intracranial, and / or tracheal administration. Additionally, antiviral nanoparticles may be administered topically rather than systemically, in depot or sustained-release formulations. Antiviral nanoparticles may be formulated with common excipients, diluents, or carriers, compressed into tablets, formulated as elixirs or solutions for convenient oral administration, or administered via intramuscular or intravenous routes. Antiviral nanoparticles may be administered transdermally and formulated into sustained-release formulations, etc. Antiviral nanoparticles may be administered alone or in combination with each other, or they may be used in combination with other known compounds. Formulations suitable for use in the present invention are found in Remington's Pharmaceutical Sciences (Mack Publishing Company (1985) Philadelphia, PA, 17th ed.), which is incorporated herein by reference. Additionally, for a brief review of drug delivery methods, refer to Langer, Science (1990) 249:1527-1533, which is incorporated herein by reference.

[0094] A sustained-release composition may be manufactured. A suitable example of a sustained-release composition comprises a semipermeable matrix of a solid hydrophobic polymer containing the antiviral nanoparticles of the present invention, said matrix existing in the form of a molded article, e.g., a film or a microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and [gamma]ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT(TM) (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0095] The antiviral nanoparticles of the present invention may also be encapsulated in microcapsules prepared, for example, by coacervation technology or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such technology is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0096] The pharmaceutical compositions described herein may be prepared in a manner known to those skilled in the art, namely by conventional mixing, dissolving, granulating, dragé-making, pulverizing, emulsifying, encapsulating, capturing, or lyophilizing methods. The following methods and excipients are merely examples and are by no means limiting. For injection, they may be formulated into a product by dissolving, suspending, or emulsifying the antiviral nanoparticles (and optionally another active agent) in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Preferably, the antiviral nanoparticles of the present invention may be formulated into an aqueous solution, preferably a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer. In the case of administration via the mucosa, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrating agents are generally known in the art.

[0097] Preferably, the pharmaceutical formulation for parenteral administration comprises an aqueous solution of a water-soluble form of antiviral nanoparticles. Additionally, the suspension of antiviral nanoparticles can be prepared as a suitable oily injectable suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. The aqueous injectable suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain a suitable stabilizer or agent that increases the solubility of the antiviral nanoparticles to enable the preparation of a highly concentrated solution.

[0098] The amount of the antiviral nanoparticles of the present invention, which can be combined with a carrier material to form a single dose, will vary depending on the viral disease being treated, the mammalian species, and the specific method of administration. Additionally, it will be understood that the specific dose level for any particular patient will vary depending on various factors, including the activity of the specific compound used; the age, weight, general health condition, gender, and diet of the individual being treated; the time and route of administration; the rate of elimination; other drugs previously administered; and the severity of the specific viral disease being treated, as is well understood by those skilled in the art.

[0099] Further embodiments of the present invention provide a method for treating and / or preventing influenza virus infection and / or influenza virus-related disease, comprising the step of administering one or more therapeutically effective amounts of the virucidal nanoparticles of the present invention to a subject in need thereof.

[0100] Another embodiment of the present invention provides the antiviral nanoparticles of the present invention for use in the treatment and / or prevention of influenza virus infection and / or diseases associated with influenza virus.

[0101] As used herein, the terms “subject” or “patient” are well-recognized in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cattle, horses, goats, sheep, pigs, camels, and, most preferably, humans. Other animals, such as chickens, are also included in these terms. In preferred embodiments, the terms “subject” or “patient” refer to humans and animals, such as dogs, cats, rats, mice, monkeys, cattle, horses, goats, sheep, pigs, camels, and chickens. In some embodiments, the subject is a subject requiring treatment or a subject infected by the influenza virus. In other embodiments, the subject may be an animal infected by avian influenza, such as a chicken. However, in other embodiments, the subject may be a healthy subject or a subject that has already received treatment. The terms do not indicate a specific age or gender. Thus, they are intended to encompass adult, child, and newborn subjects, whether male or female.

[0102] “Treatment” refers to both therapeutic treatment and preventive or protective measures. Subjects requiring treatment include subjects already infected by the influenza virus, as well as subjects for whom influenza viral infection must be prevented. Accordingly, mammals to be treated in this specification, preferably humans, may have been diagnosed as infected by the influenza virus, or may be susceptible or sensitive to the influenza virus. Treatment includes improving at least one symptom of a disease or condition caused by an influenza viral infection, curing such disease or condition, and / or preventing the occurrence of such disease or condition. Prevention means weakening or reducing the ability of the influenza virus to cause infection or disease, for example, by influencing the post-entry viral event.

[0103] "Mammals" for therapeutic purposes refer to any animal classified as a mammal, including humans, livestock and farm animals or pets, such as dogs, horses, cats, cattle, monkeys, etc. Preferably, the mammal is a human.

[0104] The term "therapeutic effective amount" refers to an amount of the antiviral nanoparticles of the present invention effective in altering the influenza virus in a recipient subject, rendering it inactive, and / or causing physiologically detectable changes in the recipient subject, for example, improving or preventing at least one symptom associated with a viral infection, or reducing the transmission rate of at least one viral substance.

[0105] Another embodiment of the present invention provides a virucidal composition comprising an effective amount of one or more virucidal nanoparticles of the present invention and optionally at least one suitable carrier or aerosol carrier. “Effective amount” refers to an amount sufficient to irreversibly inhibit the influenza virus, i.e., an amount sufficient to obtain a virucidal effect. In one embodiment, the suitable carrier is selected from the group comprising stabilizers, fragrances, coloring agents, emulsifiers, thickeners, wetting agents, or mixtures thereof. In another embodiment, the virucidal composition may exist in the form of a liquid, gel, foam, spray, or emulsion. In further embodiments, the virucidal composition may be an air freshener, a sterile solution, or a disinfectant solution.

[0106] Another embodiment of the present invention provides an apparatus (or product) comprising a viscous composition of the present invention or one or more viscous nanoparticles of the present invention and means for applying and / or dispensing the viscous nanoparticles of the present invention. In another embodiment, the means comprises a dispenser, a spray applicator, or a solid support soaked with the viscous nanoparticles of the present invention. In another embodiment, the support is a fabric or non-fabric, a textile, a paper towel, cotton wool, and an adsorbent polymer sheet, or a sponge.

[0107] Another embodiment of the present invention provides a method for disinfecting and / or sterilizing using the antiviral nanoparticles of the present invention, the antiviral composition of the present invention, or the pharmaceutical composition of the present invention.

[0108] In a preferred embodiment, a disinfection and / or sterilization method comprises: (i) providing at least one virucidal nanoparticle of the present invention, a virucidal composition of the present invention, or a pharmaceutical composition of the present invention; and (ii) contacting an influenza virus-contaminated surface or a surface suspected of being contaminated by an influenza virus with at least one virucidal nanoparticle of the present invention, a virucidal composition of the present invention, or a pharmaceutical composition of the present invention for a time sufficient to obtain a virucidal effect. In some embodiments, the influenza virus-contaminated surface is human or animal skin. In other embodiments, the influenza virus-contaminated surface is a non-biological surface, such as medical equipment, clothing, masks, furniture, rooms, etc.

[0109] Another embodiment of the present invention provides the use of the virucidal nanoparticles of the present invention, the virucidal composition of the present invention, or the pharmaceutical composition of the present invention for sterilization and / or disinfection. In some embodiments, sterilization and disinfection are on an influenza virus-contaminated surface or a surface suspected of being contaminated by the influenza virus. In some preferred embodiments, the surface is human or animal skin. In other preferred embodiments, the surface is a non-biological surface, such as medical equipment, clothing, masks, furniture, rooms, etc. In one embodiment, the virucidal composition of the present invention or the pharmaceutical composition of the present invention is used as a virucidal hand sanitizer for frequent use. In another embodiment, the virucidal composition of the present invention or the pharmaceutical composition of the present invention is applied by spraying. In a further embodiment, the virucidal composition of the present invention or the pharmaceutical composition of the present invention is applied onto a protective mask.

[0110] Those skilled in the art will recognize that the invention described herein is open to modifications and variations other than those specifically described. It will be understood that the invention includes all such modifications and variations without departing from its spirit or essential features. The invention includes all steps, features, compositions, and compounds referred to or indicated individually or collectively in this specification, as well as any two or more of said steps or features or any combination thereof. Accordingly, the present disclosure is to be illustrative rather than limiting in all embodiments, and the scope of the invention is defined by the appended claims, which are intended to include all variations within the equivalent meaning and scope.

[0111] The following description will be more fully understood by referring to the following examples. However, these examples are intended to illustrate methods for carrying out the invention and are not intended to limit the scope of the invention.

[0112] Examples

[0113] Experimental data

[0114] Density analysis

[0115] To determine the optimal 6'SLN density, NPs with three different 6-sialyl-N-acetylactosamine (6'SLN) densities were synthesized (Fig. 1). Control experiments were also performed using PEG (5) and 3-sialyl-N-acetylactosamine (3'SLN)-coated gold NPs.

[0116] A dose-response comparative study was performed on two influenza A strains, H1N1 (Neth / 09) and H3N2 (Sing / 04), and one B strain, Yamagata. LD-6'SLN NP was at the lowest half-inhibitory concentration (EC6) for all tested viruses. 50 It has ). In general, NP inhibited influenza A subtypes better than B subtypes.

[0117] In vitro half inhibition of different NPs against H1N1 Neth / 09 strain (EC 50 ) and cytotoxicity concentration (CC 50 The molar concentration was calculated separately based on NP and trisaccharide / NP. Influenza strain EC 50 (㎍ / ㎖) EC 50 NPs (nM) EC 50 6'SLN (nM) CC 50 (㎍ / ㎖) LD 6'SLN NPs(18 6'SLN / NP) H1N1 Neth / 09 0.13 0.64 11.5 >500 HD 6'SLN NPs(38 6'SLN / NP) H1N1 Neth / 09 0.45 2.2 83 > 500 LD(-) 6'SLN NPs(8 6'SLN / NP) H1N1 Neth / 09 1.1 5.4 43 > 500 LD 3'SLN NPs(18 3'SLN / NP) H1N1 Neth / 09 5.3 27.2 489 >500 PEG5 NPs H1N1Neth / 09 N / A N / A N / A > 500

[0118] EC of different NPs for H3N2 Sing / 04 strain 50 and CC 50 . Influenza strain EC 50 (㎍ / ㎖) EC 50 NPs (nM) EC 50 6'SLN (nM) CC 50 (㎍ / ㎖) LD 6'SLN NPs(18 6'SLN / NP) H3N2 SING / 04 0.58 2.8 50.4 >500 HD 6'SLN NPs(38 6'SLN / NP) H3N2 SING / 04 1.12 5.9 224.4 > 500 LD(-) 6'SLN NPs(8 6'SLN / NP) H3N2 SING / 04 1.9 9.4 75.2 > 500 LD 3'SLN NPs(18 3'SLN / NP) H3N2 SING / 04 4.5 22 396 >500 PEG5 NPs H3N2 SING / 04 N / A N / A N / A > 500

[0119] EC of different NPs against Influenza B / Yamagata 50 and CC 50 . Influenza strain EC 50 (㎍ / ㎖) EC 50 NPs (nM) EC 50 6'SLN (nM) CC 50 (㎍ / ㎖) LD 6'SLN NPs(18 6'SLN / NP) B / Yamagata 13.7 67 1206 >500 HD 6'SLN NPs(38 6'SLN / NP) B / Yamagata 17.25 85 3230 > 500 LD(-) 6'SLN NPs(8 6'SLN / NP) B / Yamagata 53.3 260 2080 > 500 LD 3'SLN NPs(18 3'SLN / NP) B / Yamagata >100 >100 >1000 >500 PEG5 NPs B / Yamagata N / A N / A N / A > 500

[0120] Subsequently, a virucidal analysis was performed to determine whether the inhibition mechanism was irreversible. In the virucidal analysis, the corresponding IC was measured over a certain period of time. 99 NPs were incubated with the virus at a given concentration. Subsequently, serial dilutions of the inoculum were performed, and the residual infectivity of the virus was measured. The viricidal activity of LD6'SLN NPs against Neth / 09 and Sing / 04 strains was tested by increasing the virus concentration tenfold compared to the dose-response experiment. The titer of the Neth / 09 strain decreased by 2 log, while that of Sing / 04 decreased by 1.5 log. A decrease of 1-2 log in viral titer indicates irreversible inhibition of the virus.

[0121] TEM study to verify virus-NP interactions

[0122] Virus-NP interactions were also demonstrated by electron microscopy (TEM). H3N2 Vic / 11 viruses were incubated with LD6'SLN NPs for 1 hour. After preparing the TEM grid, methyl tungsten staining was performed. Most of the viruses were completely covered with LD6'SLN NPs (Fig. 2b). A control experiment was performed with PEG(5) NPs, and although the NPs were all present, they did not attach to the viral envelope (Fig. 2c).

[0123] Potential of NP to inhibit avian influenza virus

[0124] Influenza epidemics generally occur when animal influenza strains are mixed with human influenza strains. Therefore, the next objective is to irreversibly inhibit avian influenza strains with the NPs of the present invention. Preliminary studies were conducted with the egg-adapted virus strain, CAL / 09. Neth / 09 and CAL / 09 are two very similar human H1N1 strains. LD-6'SLN NPs exhibit strong activity against the Neth / 09 strain. However, the CAL / 09 strain, cloned using eggs, binds to LD-3'SLN NPs with higher affinity (Table 4). These results indicate that LD-3'SLN NPs inhibit avian influenza strains.

[0125] Egg-adapted virus strains prefer -2,3 linkages, whereas mouse-adapted strains prefer -2,6 linkages. Neth / 09 mouse adapted EC 50 (㎍ / ㎖) CAL / 09 Al Adapted EC 50 (㎍ / ㎖) LD-6'SLN 0.13 34.9 LD-3'SLN 5.13 1.18

[0126] From gold core to cyclodextrin core

[0127] Human influenza virus was irreversibly inhibited by C15-6'SLN grafted gold NPs. However, for pharmaceutical applications, it is important to change the gold core to an organic material. Among different organic materials such as polymers, dendrimers, and dendrons, cyclodextrin (CD) is a preferred core for grafting ligands containing 6'SLN to target HA.

[0128] The size of the CD (d–1.5 nm) is similar to the gold (metal) NP (–3 nm) of the present invention and matches well with the HA spherical head (–5 nm). Similar to the gold (metal) NP, the cyclodextrin has a hard chemical structure that contributes to antiviral activity in conjunction with the ligand. Additionally, the β-cyclodextrin can have up to 7 trisaccharides (more preferably 3 or 4 trisaccharides), and 3 is the exact number of sialic acid binding sites within the HA spherical head (Fig. 3a). Thus, the β-CD was modified into a C15-6'SLN in a manner very similar to that of gold nanoparticles.

[0129] Compared to previous organic materials containing sialic acid, significantly lower EC against different human influenza viruses 50 Values ​​were obtained (Fig. 3b). The antiviral activity of the modified cyclodextrin was demonstrated in both in vivo and in vitro experiments, in which the viral titer was reduced by several logarithms (Figs. 3c and d). The successful results in vitro indicate that the modified cyclodextrin will irreversibly inhibit the virus in vivo.

[0130] This specification demonstrates that the selection of ligands, as well as trisaccharides, is critical for the irreversible inhibition of the human influenza virus with nanomaterials. C15-6'SLN ligands on two different cores, gold nanoparticles and cyclodextrin, inhibited various strains of the human influenza virus at very low nanomaterial concentrations. Compared to trisaccharide units, EC10 in the low nM range (1–100 nM) 50 While the concentration was achieved, the literature EC 50 The value was 50 to 500 times higher than that of similar substances.

[0131] In vitro antiviral activity of modified cyclodextrin

[0132] To investigate the relationship between chemical structure and antiviral activity, β-cyclodextrin (β-CD) was modified with different ligands, with or without trisaccharides. Exemplary modified cyclodextrins are shown in Fig. 4. These 1 It contains a similar number of trisaccharides as measured by H nuclear magnetic resonance spectroscopy (NMR) (see Fig. 5 and Table 5). The inhibitory activities of these NPs were compared by performing dose-response analyses against the influenza A / Netherlands / 2009 (H1N1) strain (A / NL / 09) (Fig. 6). Infection was quantified by immunocytochemical analysis at 24 hours post-infection (hpi). Sufficiently long, hydrophobic ligands and 6'SLN terminal groups ( C6-6'SLN, C11-6'SLN and C14-6'SLN β-CD containing ) exhibited strong inhibitory activity against cell infection by influenza A / NL / 09, and EC in the nanomolar range 50 It has a value. On the other hand, shorter ligands, C1-6'SLN β-CDs containing [subject] did not effectively inhibit infection. Introducing a sufficiently long ligand clearly improved terminal-group flexibility; consequently, the inhibitory concentration decreased. EC 50 The hydrophobic ligand is a hydrophilic PEG8 ligand ( PEG8-6'SLN It was similar (but slightly higher) when replaced with ).

[0133] 1 Average number of 6'SLNs or 3'SLNs per β-cyclodextrin and corresponding molecular weight determined by 1H NMR. Number of spacers / CD Number of 6'SLN / CD Molecular weight (kDa) P8-6' 4 3.5 5.7 C14-6' 3.1 3 4.1 C11-6' 3.6 3.1 4.1 C6-6' 2.8 2.7 3.4 C11-3' 3 2.7 3.9 C1-6' - 3 3.5

[0134] Nanoparticles that showed the best inhibitory activity against A / NL / 09, C11-6'SLNIt exhibited strong antiviral activity against human influenza strains of both types A and B (Table 6 and Figure 7). Importantly, it inhibited very recent clinical A(H1N1) and B strains (from the 2017 / 2018 influenza season) that were isolated from patients at the University Hospital in Geneva and passaged only once in cells. C11-6'SLN It did not show any antiviral activity against HSV-2, an HSPG-binding virus, which indicates the specificity of the compound against sialic acid-dependent viruses.

[0135] While the 6'SLN is known to be specific to human influenza strains, the 3'SLN is preferred as a primary attachment site for avian influenza strains. In particular, to demonstrate the generality of this approach for influenza strains known to be capable of crossing the species barrier, C11-3'SLN Synthesized and tested against avian influenza strains (Fig. 4). C11-3'SLN It successfully inhibited two avian strains, H5N1 and H7N1, at concentrations of 4.1 and 8.8 µg / ml, respectively (see Table 6). In fact, these results confirm the strategy adopted against human strains. Importantly, one of these avian strains, H5N1, has a significant potential to cause the next influenza pandemic. C11-3'SLN Whether this human strain, A / NL / 09, can be inhibited and C11-6'SLN We further tested whether it has activity against this algal strain, H5N1. C11-3'SLN While [it] showed excellent inhibitory activity against A / NL / 09 (Fig. 4 and Table 6), C11-6'SLNIt showed no activity against H5N1 (Table 6 and Figure 8). These results are similar to previous literature comparing the binding affinities of avian and human strains for different types of sialic acid. Avian influenza strains (particularly H5N1 strains) preferentially bind to alpha-2,3-linked sialic acid, which has a thin, straight trans form. On the other hand, the broader sialic acid binding sites of human strains can accommodate both the bulky cis form of alpha-2,6-linked sialic acid and the narrower -2,3-linked sialic acid.

[0136] Inhibitory activity of C11-6'SLN and C11-3'SLN against different influenza strains. compound CC 50 (㎍ / ㎖) EC 50 (㎍ / ㎖) EC 50 † (nM) A / Netherlands / 2009 (H1N1) C11-6' >100 0.18 (0.14 - 0.24) 42 C11-3' >100 6.5 (4.1-10.1) >1000 A / Clinical / 2018 (H1N1) C11-6' >100 0.5 (0.4 - 0.67) 125 Singapore / 2004 (H3N2) C11-6' >100 0.23 (0.16 - 0.34) 56.5 B / Wisconsin / 2010* C11-6' >100 2.2 (1.49 - 3.42) 500 B / Clinical / 2018 C11-6' >100 20 (10.5 - 28.7) >1000 A / turkey / Turkey / 2005 (H5N1) C11-3' >100 4.1 (2.55-6.7) 931 C11-6' >100 N / A N / A A / turkey / Italy / 1999 (H7N1) C11-3' >100 8.8 (3.2-26) >1000 HSV-2 (Control group) C11-6' >100 N / A N / A

[0137] * B Yamagata subtype

[0138] † Molar concentration was determined based on the number of cyclodextrin cores.

[0139] CC 50 : Half maximum cytotoxic concentration.

[0140] Next, a virucidal analysis was performed to determine the mechanism of inhibition, namely virucidal (irreversible) or viral inhibitory activity (reversible). The synthesis of similar nanoparticles sharing a β-cyclodextrin core and a 6'SLN moiety but with different ligands highlights the structural features conferring virucidal activity (Figs. 9 and 10). It was hypothesized that one of the key elements of irreversible viral inhibition is that the binding moiety (here, the 6'SLN) is contained by a hydrophobic ligand. To verify this hypothesis, C11-6'SLN and P8-6'SLN ...was compared. Simply put, the amount of compound providing complete protection (10 μg of C11-6' and 50 μg of P8-6' ) was incubated with A / NL / 09 for 1 hour. Infectivity was evaluated after performing serial dilutions of the inoculum. C11-6' In the case of ( aThe graph of ) shows that complete protection was maintained upon dilution and ( c The graph of ) shows that this characteristic has been found for several different strains. This is called the irreversible (i.e., resistance to dilution) inhibitory activity antiviral mechanism. P8-6' In the case of, ( b The graph shows that while complete protection existed at the initial concentration, the difference in infectivity compared to the control sample (virus alone) was lost upon dilution; in other words, the inhibitory effect was found to be reversible (viral inhibition). These two nanoparticles exhibit similar inhibitory activity, differing only in the hydrophobicity of their ligands. In the antiviral analysis, C11-6'SLN While silver reduced the viral titer by 1,000 times (Fig. 9b), P8-6'SLN In this case, the infection was completely recovered (Fig. 9a). Therefore C11-6'SLN While it has an irreversible inhibitory effect on viruses, P8-6'SLN The effect is reversible. It is worth noting that both nanoparticles are non-toxic to cells (Figs. 9a and 9b). Against other influenza strains C11-6'SLN The antiviral activity of was further investigated, and its irreversible activity independent of the strain was confirmed (Fig. 9c).

[0141] In vitro activity of modified cyclodextrin

[0142] 3D model of reconstructed human airway epithelium, MercilAir ® In vitro experiments were performed. These air-liquid interface cultures perfectly mimic the pseudostratified structure (basal, ciliated, and goblet cells) and barrier defense mechanisms (i.e., mucociliary clearance and epithelial cell immunity) of the human upper respiratory epithelium, which are the major sites of influenza virus replication in humans. In vitro experiments were performed using clinical H1N1 epidemic 09 strains that had not been passaged in cells to exclude any adaptive bias. C11-6'SLN or P8-6'SLN (50 µg / tissue) and virus (10 4RNA copies (tissue) were first simultaneously added to the apical surface of the tissue without prior incubation. After 4 hours, the inoculum was removed, the tissue was washed, and the progression of infection was monitored daily by qPCR from the tissue apical wash without any re-addition of nanoparticles. C11-6'SLN While completely preventing virus replication throughout the entire experiment, P8-6'SLN It slightly reduced viral replication during the first 2 days (dpi) after infection, but did not thereafter (Fig. 11a).

[0143] also, C11-6'SLN In tissues treated with, the inhibition of viral replication was reflected in the absence of infected cells and the original morphology of the treated tissue, which is the same as in untreated tissue or P8-6'SLN - It was significantly different from the treated tissue (Fig. 11b). The lack of lactate dehydrogenase (LDH) release in immunofluorescence imaging and apical washing demonstrated that not only the ciliated cell layer but also physiological ciliary movement and tissue integrity were preserved (Figs. 11b and 12). In complete contrast, the untreated tissue or P8-6'SLN - The treated control group showed a decrease in thickness due to changes in the ciliated cell layer and the presence of infected cells (Fig. 11b). To rule out infectious residual virus levels detected by qPCR in the treated tissues, the tissues were maintained in culture for 23 days; however, no increase in viral titers was observed over time, whereas the untreated tissues were continuously shedding the virus (Fig. 13). Importantly, starting at 1 dpi to mimic therapeutic administration, C11-6'SLN In vitro experiments were also performed under stricter post-treatment conditions, with (30 µg / tissue) administered every 24 hours for 4 days. Furthermore, under these conditions, the nanoparticles exhibited significant inhibitory activity, demonstrating their potential as a therapeutic agent (Fig. 11c). In the same in vitro model, high doses administered daily C11-6'SLNThe biocompatibility of was evaluated. C11-6'SLN It did not exhibit any cytotoxic or pro-inflammatory activity under the conditions described above (Fig. 14).

[0144] In vivo activity of modified cyclodextrin

[0145] In vivo experiments were performed using BALB / c mice under both co-treatment and post-treatment conditions. In the co-treatment experiments, the mice were given C11-6'SLN (25 µg / mouse) and A / NL / 09 (100 infectious particles / mouse) were administered simultaneously via the intranasal route. C11-6'SLN To estimate the effect of administration on the physiological status of infected animals, the body temperature and body weight of mice were measured on a daily basis. At 2 dpi, half of the mice were randomly euthanized and the remaining mice C11-6'SLN They were retreated. Mice in the second group were euthanized at 4 dpi. Viral titers were quantified by bronchoalveolar lavage (BAL) (Fig. 15a). Significant reductions in viral titers were observed in treated mice at 2 and 4 days post-infection (Fig. 15a). C11-6'SLN The antiviral activity of also significantly reduced morbidity, along with substantial preservation of both body weight and body temperature compared to untreated mice (Figs. 15b and 15c). Collectively, these results C11-6'SLN Nanoparticles demonstrate the ability to prevent in vivo infection and the spread of viruses in the lungs.

[0146] C11-6'SLN The in vivo therapeutic potential was evaluated through post-treatment conditions. Mice were infected with A / NL / 09 (100 infectious particles / mouse) and then treated daily for 3 days. C11-6'SLN They were treated with the same dose of nanoparticles at 6 hpi (14 µg / mouse) (Figs. 15d–f). The body weight and body temperature of the mice were measured daily. C11-6'SLNAlthough less potent under post-treatment conditions, it still delayed the progression of infection. Treated mice showed reduced signs of morbidity (Figs. 15d and 15e) and better clinical scores. This improvement in the physiological status of infected animals is associated with long-term survival (Fig. 15f).

[0147] method

[0148] Synthesis of modified cyclodextrin

[0149] chemical Neu5Acα(2,6)-Galβ(1-4)-GlcNAc-β-ethylamine and Neu5Acα(2,3)-Galβ(1-4)-GlcNAc-β-ethylamine were purchased from TCI Chemicals. Heptakis-(6-deoxy-6-mercapto)-beta-cyclodextrin and carboxymethyl-beta-cyclodextrin sodium salt were purchased from Cyclodextrin-Shop. 11-Dodecenoic acid was purchased from abcr GmbH. 14-Pentadecenic acid was purchased from Larodan AB. Maleimide-PEG8-CH2CH2COOH was purchased from PurePEG. All other chemicals and solvents were purchased from Sigma-Aldrich.

[0150] method Cyclodextrin derivatives targeting the influenza virus were synthesized in three steps. The first step was conjugating a ligand to the cyclodextrin. The second step was N-hydroxysuccinimide (NHS) activation of the -COOH terminal group of the ligand. The third step was grafting SLN onto the ligand.

[0151] Step 1: Modification of β-cyclodextrin as a ligand

[0152] 0.04 mmol of heptakis-(6-deoxy-6-mercapto)-beta-cyclodextrin was stirred overnight in the presence of a UV lamp (250 W) with 0.28 mmol of a bifunctional molecule (ligand) containing allyl and carboxylic acid terminal groups (e.g., 6-heptene, 11-dodecenoic acid, or 14-pentadecenic acid) in 5 mL of DMSO. The resulting heptakis-(6-deoxy-6-alkanexanthione)-beta-cyclodextrin derivative was precipitated from the DCM-diethyl ether mixture by centrifugation and dried under vacuum.

[0153] To modify β-cyclodextrin with a PEG8 spacer, 0.04 mmol heptakis-(6-deoxy-6-mercapto)-beta-cyclodextrin was stirred overnight with 0.28 mmol maleimide-PEG8-CH2CH2COOH in 5 mL of phosphate buffer (pH: 6.8). The modified β-cyclodextrin was purified by dialysis and dried by lyophilization.

[0154] Step 2: NHS Activation Response

[0155] 0.04 mmol of the cyclodextrin derivative obtained in Step 1 was stirred overnight with 1.12 mmol of N-hydroxysuccinimide (NHS), 0.56 mmol of ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC-HCl), and 0.02 mmol of 4-dimethylaminopyridine (DMAP), respectively, in 5 mL of DMSO. The resulting NHS-activated cyclodextrin derivative was first precipitated in ice-cold water and then washed with acetonitrile and diethyl ether. The product was dried under vacuum.

[0156] To obtain C1-6', Step 1 was omitted and the NHS activation reaction was performed directly. Carboxymethyl-beta-cyclodextrin sodium salt (0.04 mmol) was directly activated with 1.12 mmol NHS and 0.56 mmol EDC-HCl, and 0.02 mmol DMAP was added. The activation reaction was carried out overnight. The resulting cyclodextrin derivative was first precipitated from a DCM-diethyl ether mixture and washed three additional times. The product was dried under vacuum. 6'SLN grafting was performed in the same manner as in Step 3.

[0157] Step 3: Trisaccharide Grafting

[0158] 5.6 μmol Neu5Acα(2,6)-Galβ(1-4)-GlcNAc-β-ethylamine (Neu5Acα(2,3)-Galβ(1-4)-GlcNAc-β-ethylamine for C11-3') was mixed with 1.6 μmol of the cyclodextrin derivative obtained in Step 2. 15 μmol of triethylamine (TEA) was added, and the reaction was carried out overnight in 1 mL of DMSO. The reaction product was diluted with 0.01 M phosphate buffer (pH: 7.5) and concentrated using an Amicon filter (MWCO: 3k). The resulting heptakis-(6-deoxy-6-SLN-ethylcarboxymido-alkylthio)-beta-cyclodextrin derivative was further washed with distilled water and dried by freeze-drying. Grafting of trisaccharides onto ligand-modified cyclodextrin is 1 This was confirmed by H and DOSY NMR studies (Figs. 16 and 17).

[0159] Synthesis of C15-6'SLN modified cyclodextrin

[0160] C15 modification of β-CD 55 mg of thiol-modified β-CD and 70 mg of 14-pentadecenic acid in 5 ml of DMSO were stirred overnight under UV light.

[0161] NHS activation of C15-β-CD: The generated material was activated overnight using 100 mg NHS, 75 mg EDC, and 2.5 mg DMAP in DMF. NHS-activated C15-β-CD was precipitated in ice-cold water and washed three additional times. The final precipitation was performed in acetonitrile.

[0162] 6'SLN grafting on C15-β-CD : 5 mg C15-β-CD, 5 mg amine-functionalized 6'SLN, and 3 mg TEA were stirred overnight in DMSO. The resulting material was purified using an Amicon filter.

[0163] Synthesis of PEG nanoparticles

[0164] Synthesis of PEG(5) NPs : 88.6 mg of tetrachloroauric acid trihydrate (HAuCl4*3H2O) in 15 ml of EtOH was mixed with 56 mg of HS-PEG (5) in 5 ml of EtOH for 10 minutes. 94.6 mg of sodium borohydrate (NaBH4) in 37.5 ml of EtOH was added dropwise to the mixture. To form complete NPs, the reaction was carried out overnight. The resulting NPs were cleaned with an Amicon filter using an EtOH-H2O solvent mixture.

[0165] PEG(5)-C15 mixed ligand NP : A ligand exchange reaction was carried out overnight using 1.5 mg of 16-mercaptohexacanoic acid (HS-C15-COOH) and 20 mg of PEG (5) NP in DMF. The NP was precipitated from the DMF-diethyl ether mixture and washed three additional times.

[0166] Activation of NHS by NPs : 15 mg of NP was activated overnight using 10 mg of N-hydroxysuccinimide ester (NHS), 2 mg of ethylcarbodiimide hydrochloride (EDC), and 0.1 mg of 4-(dimethylamino)-pyridine (DMAP) in DMF. The resulting NP was precipitated from a DMF-diethyl ether mixture and washed three additional times.

[0167] 6'SLN grafting on NP : 5 mg of NHS-activated NP in 1 ml of DMSO was mixed with 1.7 mg of amine-functionalized 6'SLN in 3 ml of 0.1 M phosphate buffer (pH: 7.5). After 5 hours of reaction, the 6'SLN-grafted NP was cleaned with an Amicon filter.

[0168] Biological analysis

[0169] substance DMEM-Glutamax medium was purchased from Thermo Fischer Scientific. Tween 20 was used as the wash buffer. ® and 3,3'-diaminobenzidine (DAB) purifications were purchased from Sigma-Aldrich. The primary antibody (influenza A monoclonal antibody) was purchased from Light Diagnostics. The secondary antibody (anti-mouse IgG, HRP-linked antibody) was Cell Signaling Technologies ® Purchased from. Celtiter 96® AQ containing an electron coupling reagent (phenazine ethosulfate; PES) and a tetrazolium compound [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, salt retardant; MTS]. ueous The One Solution Cell Proliferation Analyzer was purchased from Promega.

[0170] Cell culture:

[0171] MDCK (Madin-Darby canine kidney cell) cell lines were purchased from ATCC (American Type Culture Collection, Rockville, MD). Cells were cultured in Dulbecco’s modified Eagle medium containing glucose supplements (DMEM+GlutaMAX™) with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. MDCK cell lines were grown at 37°C in a humid atmosphere with CO2 (5%).

[0172] Virus strain:

[0173] Clinical isolates of HSV-2 were originally provided by Professor M. Pistello (University of Pisa, Italy) and propagated by plaque analysis on Vero cells and was titrated. H1N1 Neth09 and B Yamagata were a kind gift from Professor M. Schmolke (University of Geneva). Algae strain NIBRG-23 (A / turkey / Turkey / 1 / 2005 H5N1 (prepared by reverse genetics using surface protein and A / PR / 8 / 34 (H1N1) backbone) creature National Institute for Standards and Control Biological Standards and Controls), obtained from Potters Bar, UK, and after birth Virus purification and characterization were performed after further growth in 10-day-old developing eggs. Clinical samples were obtained from anonymized patients at the University Hospital of Geneva. All influenza strains were propagated and titrated by ICC against MDCK cells in the presence of TPCK-treated trypsin (0.2 mg / ml).

[0174] Cell viability analysis

[0175] Cell viability was measured by the MTS [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Confluent cell cultures dispensed into 96-well plates were triple-incubated with different concentrations of nanoparticles or ligands under the same experimental conditions described for the antiviral assay. Cell viability was determined by the Celltiter 96 proliferation assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Absorbance was measured at 490 nm using a microplate reader (Model 680, BIOSARD). The effect of different concentrations of nanoparticles or cyclodextrin on cell viability was expressed as a percentage by comparing the absorbance of treated cells to that of cells incubated with culture medium alone. 50% cytotoxicity concentration (CC 50 The ) and 95% confidence interval (CI) were determined using PRISM software (Graph-Pad software, San Diego, CA).

[0176] Suppression analysis

[0177] MDCK cells were pre-squashed in a 96-well plate 24 hours prior. After incubating with increasing concentrations of the substance with influenza virus (MOI: 0.02 or 0.01 for H5N1 and 0.1 for other viruses) at 37°C for 1 hour, the mixture was added to the cells. After virus adsorption (1 hour at 37°C), the viral inoculum was removed, the cells were washed, and fresh medium was added. After incubating at 37°C for 24 hours, infection was analyzed by immunocytochemistry (ICC). The cells were fixed and permeated with methanol. Subsequently, the primary antibody (1:100 dilution) was added, and the cells were incubated at 37°C for 1 hour. The cells were washed three times with wash buffer (DPBS + Tween 0.05%); then, the secondary antibody (1:750 dilution) was added. After 1 hour, the cells were washed, and DAB solution was added. Infected cells were counted, and the percentage of infection was calculated by comparing the number of infected cells under treated and untreated conditions.

[0178] For H5N1, flow cytometry-based inhibition analysis was additionally performed. MDCK cells were pre-squashed in 24-well plates (75,000 cells / well) 24 hours prior. After incubating with increasing concentrations of the substance with influenza virus (MOI: 0.04) at 37°C for 1 hour, the mixture was added to the cells. After virus adsorption (1 hour at 37°C), the viral inoculum was removed, the cells were washed, and fresh medium was added. After incubating at 37°C for 5 hours, infection was analyzed by flow cytometry. Briefly, cells were trypsinized and fixed using IC fixation buffer (Thermo Fischer Scientific, Netherlands) for 15 minutes at room temperature, and then permeated using 1X IC permeation buffer (Thermo Fischer Scientific, Netherlands) for 15 minutes at 4°C. Cells were stained with the anti-influenza A virus nucleoprotein mouse monoclonal antibody [D67J] (FITC) (Apkem ab210526, Netherlands) for 30 minutes at 4°C. The antibody dilution was 1:80 in permeation buffer, and 50 µl was added per tube. FACS analysis was performed using FACS Calibre 3 software. The concentration that reduces the number of infected cells by 50% (effective concentration (EC10)) 50 )) was determined using Prism software. The gating strategy for FACS was performed as shown in Fig. 18. After performing the first gating based on FSC / SSC, a negative gating was created for FITC. This gating was applied to the remaining samples.

[0179] H7N1 infectivity was evaluated through luciferase activity. MDCK cells were placed in 5×10⁶ well plates. 4The cells were aliquoted. After 24 hours, the medium was replaced with serum-free medium. Incubation was performed with 100 pfu of H7N1 A / Turkey / Italy / 977 / 1999 encoding increasing concentrations of C11-3' nanoluciferase. The mixture was incubated at 37°C for 1 hour, then added to the cells and incubated for another 1 hour at 37°C (100 µl per well). The cells were washed, and the medium was replaced with serum-free medium containing 1 µg / ml TPCK-trypsin. 24 hours after infection, the cells were washed, and a 1 / 2 diluted nanoglo in PBS was added. ® It was dissolved at 40 µl per well in luciferase assay buffer (Promega). Luciferase activity was measured in cell lysates using a Tekane Infinite M200PRO plate reader: 1 / 5000 diluted Nanoglo in PBS ® 15 µl of luciferase assay substrate (Promega) was added to 15 µl of lysate for each well.

[0180] Salvia analysis

[0181] Virus (Focus-forming unit (ffu): 10 5 / ㎖) and test substance (EC 99 The concentration (Table 7) was incubated at 37°C for 1 hour. Serial dilutions of the virus-material complex were performed with an untreated control group and transferred to cells. After 1 hour, the mixture was removed and fresh medium was added. The next day, the viral titer was evaluated by ICC analysis. For the ICC analysis, the same procedure as described above was followed.

[0182] Concentration of the substance on which in vitro virus analysis was performed. substance Concentration (µg / ml) A / Netherlands / 2009 H1N1 C6-6' 100 C11-6' 100 C14-6' 100 P8-6' 500 C11-3' 500 NPs C15-6'SLN 100 NPs PEG4-6'SLN 500 A / Singapore / 2004 H3N2 C11-6' 100 B / Wisconsin / 2010 C11-6' 200

[0183] Data analysis

[0184] All results are presented as the average values ​​of three independent experiments performed with overlap. EC for the inhibition curve 50Values ​​were calculated by regression analysis using the GraphPad Prism version 5.0 program (GraphPad Software, San Diego, California, USA) and a variable slope-sigmoid dose-response curve was fitted.

[0185] In vitro

[0186] Co-treatment No. 1: H1N1 Neth / 09 strain (pfu: 10 4 ~10 5 ) and CD-C15-6'SLN (400 µg / ml) were simultaneously added to Mercil Air (reproduction of human airway epithelium). After 4 hours, the supernatant was removed and fresh medium was added. The viral titer in the supernatant was tracked every 24 hours by qPCR.

[0187] Co-processing No. 2: Mercile Air tissue, human airway epithelium reproduced in vitro (Epitellix (Geneva, Switzerland) was cultured at an air-liquid interface in a mixture of nasal polyp epithelial cells derived from healthy donors. Influenza H1N1 pdm 2009 clinical strains (1e4 RNA copies / tissue) and C11-6' (50 µg / tissue) were transferred to tissues without pre-incubation, along with untreated controls. After 4 hours of incubation, the tissues were washed twice. The basal medium was changed daily. To perform daily qPCR measurements, 200 µl of medium was added to the tissues and collected after 20 minutes. RNA extracted with the EZNA virus extraction kit (Omega Biotech) was quantified using qPCR with the Quantitech kit (#204443; Qiagen, Hilden, Germany) on a Step One ABI thermocycler.

[0188] Post-treatment: Mercil-Air tissues were infected with H1N1 pdm 2009 (1e4 copy / tissue). After 4 hours, the inoculum was removed and the tissues were washed. After 20 hours, a 20' peak wash was performed, followed by the addition of C11-6' (30 µg / tissue) or an equal volume of medium from the untreated tissue to the peaks. Fresh addition of C11-6' was performed daily after the 20' peak wash. Subsequently, RNA was extracted and qPCR was performed as described above. For toxicity studies, a similar procedure was performed in the absence of the virus.

[0189] Immunofluorescence

[0190] Influenza-infected cells were directly detected with influenza A antibodies (Right Diagnostics), and beta-tubulin primary rabbit antibodies (ApkAM) were used as markers for ciliated cells. Alexa 488-goat anti-rabbit Ab and Alexa 594-goat anti-mouse Ab (Life Technologies) were used as secondary antibodies, and nuclei were stained with DAPI. Images were acquired using a Zeiss LSM 700 meta-confocal microscope and processed with Imaris.

[0191] Lactate Dehydrogenase Analysis (LDH)

[0192] LDH release from the basal medium was measured using a cytotoxicity detection kit (Roche 04744926001).

[0193] MTT analysis

[0194] MTT solution was diluted with Mercil Air medium (1 mg / mL), and 300 µL was added as a base to a 24-well plate. After incubation at 37°C for 4 hours, the tissues were transferred to new plates and lysed with 1 mL of DMSO. The supernatant was read at 570 nm. The percentage of viability was calculated by comparing the treated tissues with the untreated tissues.

[0195] ELISA

[0196] Interleukin-6 (IL-6), CXC motif chemokine 10 (CXCL10 or IP-10), CC motif chemokine 5 (CCL5 or RANTES), interleukin-8 (IL-8 or CXCL-8), and interferon lambda (IL-29 / IL-28B) were measured in basal medium by ELISA (R&D DY206-05, DY266-05, DY278-05, DY208-05, and DY1598B-05) after daily treatment with various concentrations of CD.

[0197] In vivo

[0198] Pre-processing : 4 groups of 5 BALB / c mice were treated on day 0 with 50 µl of PBS or C11-6' (25 µg in 50 µl) and immediately treated with A / NL / 09 (10 2 They were inoculated with ffu. Body temperature and body weight of the mice were measured daily. Two days after infection, two groups of mice were sacrificed (one group treated with pbs and the other with C11-6'). Lung homogenates, nasal mucosa, and bronchoalveolar lavages were collected, and viral titers were quantified via qPCR. The C11-6' treated group was re-treated with the same amount of nanoparticles. Two days later, all remaining mice were sacrificed, and lung homogenates and nasal mucosa were collected. After tissue destruction, RNA was extracted with Trizol and quantified using qPCR, while BAL performed plaque analysis. Two independent experiments were conducted.

[0199] Post-processing : Two groups of 10 BALB / c mice A / NL / 09(10 2Mice were infected with ffu and treated 6 hours after infection, followed by daily treatment for 7 days. Body temperature and body weight were measured daily. A human endpoint was used during the survival study: mice were euthanized for uterine dislocation when body weight decreased to 75% of their starting weight. Additionally, animals that reached a necrotic state (unresponsive and unable to perceive stimuli) were also euthanized.

[0200] Dose-response analysis

[0201] Fixed virus concentration (ffu: 10 3 ) was incubated with various doses of nanoparticles at 37°C for 1 hour. Afterward, the mixture was transferred to cells. After 1 hour, the mixture was removed and the cells were washed. The next day, infection was analyzed by immunocytochemistry (ICC).

[0202] References

[0203] 1. Mammen, M., Choi, S.-K. & Whitesides, GM Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew. Chem. Int. Ed. 37, 2754-2794 (1998).

[0204] 2. Reuter, J.D. et al. Inhibition of Viral Adhesion and Infection by Sialic-Acid-Conjugated Dendritic Polymers. Bioconjug. Chem. 10, 271-278 (1999).

[0205] 3. Papp, I. et al. Inhibition of Influenza Virus Infection by Multivalent Sialic-Acid-Functionalized Gold Nanoparticles. Small 6, (2010).

[0206] 4. Papp, I. et al. Inhibition of Influenza Virus Activity by Multivalent Glycoarchitectures with Matched Sizes. ChemBioChem 12, 887-895 (2011).

[0207] 5. Tang, S. et al. Antiviral Agents from Multivalent Presentation of Sialyl Oligosaccharides on Brush Polymers. ACS Macro Lett. 5, 413-418 (2016).

[0208] 6. Kwon, S.-J. et al. Nanostructured glycan architecture is important in the inhibition of influenza A virus infection. Nat. Nanotechnol. 12, 48-54 (2017).

Claims

Claim 1 A virucidal nanoparticle comprising a core and a plurality of ligands covalently connected to the core via -S- or -O- groups, wherein at least a portion of the ligands comprises a trisaccharide moiety, wherein:- the core is a beta-cyclodextrin and has up to eight ligands comprising the trisaccharide moiety,- the ligands are hydrophobic and substituted or unsubstituted C6-C 15 An antiviral nanoparticle comprising an alkyl ligand, wherein the alkyl ligand is a polyethylene glycol (PEG) ligand or a carboxyalkyl ligand; each trisaccharide moiety is selected from 3-sialyl-N-acetylactosamine (3'SLN) and 6-sialyl-N-acetylactosamine (6'SLN); and the percentage ratio between a ligand not containing a trisaccharide moiety and a ligand containing a trisaccharide moiety is in the range of 75:25 to 95:

5. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 Antiviral nanoparticles represented by formula (II) or pharmaceutically acceptable salts thereof: Here: each R is independently hydrophobic and is a substituted or unsubstituted C6-C that is covalently bonded to a glucopyranosid unit via an -S- or -O- group. 15 It is an alkyl ligand, wherein the alkyl ligand is a polyethylene glycol (PEG) ligand or a carboxyalkyl ligand, wherein at least two of the ligands have a trisaccharide moiety selected from the group comprising 3-sialyl-N-acetyllactosamine (3'SLN) and 6-sialyl-N-acetyllactosamine (6'SLN), or at least one of the ligands has 3'SLN and the other has 6'SLN; each R' is independently H, -SO3 - , -SO4 - , -(CH2) y -COOH, -(CH2) y -SO3 - , or -(CH2) y -SO4 - and; x is 6, 7 or 8; and y are integers from 4 to 20. Claim 10 In claim 9, R' is a virucidal nanoparticle that is H. Claim 11 delete Claim 12 delete Claim 13 A pharmaceutical composition for treating and / or preventing influenza virus infection, comprising an effective amount of one or more antiviral nanoparticles of any one of claims 1, 9 and 10 and at least one pharmaceutically acceptable excipient, carrier and / or diluent. Claim 14 A pharmaceutical composition according to claim 13, wherein the carrier is an aerosol carrier. Claim 15 The pharmaceutical composition of claim 13 for use in disinfection and / or sterilization. Claim 16 An apparatus comprising one or more viscous nanoparticles of any one of claims 1, 9 and 10, and a viscous composition or means for applying or dispensing said viscous nanoparticles. Claim 17 delete Claim 18 delete

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