WO2019135715A1 - Nanoparticules hybrides de lipide-polymère - Google Patents

Nanoparticules hybrides de lipide-polymère Download PDF

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Publication number
WO2019135715A1
WO2019135715A1 PCT/SG2019/050009 SG2019050009W WO2019135715A1 WO 2019135715 A1 WO2019135715 A1 WO 2019135715A1 SG 2019050009 W SG2019050009 W SG 2019050009W WO 2019135715 A1 WO2019135715 A1 WO 2019135715A1
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lipid
composition according
nanoparticles
composition
plga
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PCT/SG2019/050009
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English (en)
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Say Chye Joachim Loo
Jongsuep BAEK
Chuan Hao TAN
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Nanyang Technological University
National University Of Singapore
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Publication of WO2019135715A1 publication Critical patent/WO2019135715A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
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    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
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    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
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    • A61K9/513—Organic macromolecular compounds; Dendrimers
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    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
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    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • This invention relates to hybrid nanoparticles for sustained release of drugs.
  • Microbial infection is emerging as one of the most deadly classes of infectious disease, due to the rapid evolution of antimicrobial-resistant strains.
  • antimicrobial resistance has led to more than 2 million infections and about 23,000 deaths each year, resulting in costs of around $20 billion in medical spending and $35 billion in lost productivity.
  • the US National Institutes of Health (NIH) have estimated that 65-80% of the microbial infections occurring in the human body are biofilm-mediated.
  • Biofilms are a dominant form of microbial life that are commonly found as structured communities of bacteria housed within a self-secreted extracellular matrix. This biopolymeric matrix provides numerous fitness benefits to the bacteria, including protection from environmental stresses, enhanced nutrient availability and increased resilience via phenotypic diversifications. Clinical studies suggest that biofilm matrix not only shields bacteria from the immune phagocytic cells, but also protects them from antibiotics by reducing the diffusion of antimicrobial agents into the biofilms. In addition, bacteria growing within a biofilm matrix can differentiate into variants with lower metabolic activities, rendering the bacterial cells less susceptible to certain antibiotics and hence, leading to a persistent infection.
  • biofilm mode of growth is not the only route used by bacterial pathogens for protection against antimicrobial agents.
  • Some pathogens such as Mycobacterium tuberculosis, Listeria monocytogenes and Salmonella enterica have evolved the capability to invade and survive inside mammalian cells, including the phagocytic cells that are generally responsible for bacterial clearance.
  • Other pathogens such as Escherichia coli and Enterococcus faecalis can reside in the Umbrella and Translational cells along the urinary tract, while Staphylococcus aureus is capable of propagating in keratinocytes. Both the biofilm and residence within a mammalian cell may be considered as a form of shielding for the microbe(s) involved.
  • microbes that display a shielding effect not only tend to cause numerous chronic and/or recurrent infections, such as urinary tract infections (UTI), pulmonary infections, endocarditis, etc., but also potentially contribute to the rapid evolution of antimicrobial-resistant strains.
  • UMI urinary tract infections
  • pulmonary infections pulmonary infections
  • endocarditis etc.
  • Gold- and silver-based nanoparticles have been used to kill the bacteria, but these nanoparticles are not able to encapsulate antibiotics.
  • Different antibiotics have differing water solubility, which is a challenge in developing antibiotic- loaded particulate systems.
  • polymer-based nanoparticles generally have low encapsulation efficiency for hydrophilic drugs, and low affinity to cellular membrane.
  • lipid-polymer nanoparticle composition for drug delivery comprising:
  • a core portion comprising a hydrophobic polymeric matrix material
  • a shell portion comprising a lipid and a surfactant
  • the lipid comprises a cationic lipid
  • the weight:weight ratio of the cationic lipid to hydrophobic polymeric matrix material is from 1 :1 to 1 :50.
  • the hydrophobic polymeric matrix material may be selected from one or more of the group consisting of poly(glycolic-co-L,D-lactic acid) (PGLA), poly(L-lactide) (PLLA), poly(s- caprolactone) (PCL), poly(glycolide) (PGA), poly(lactide) (PLA), alginate, chitosan, polysaccharide, hyaluronic acid, and co-polymers thereof (e.g. the hydrophobic polymeric matrix material is poly(glycolic-co-L,D-lactic acid) (PGLA);
  • the composition may further comprise a phospholipid conjugated to polyethylene glycol distributed in the shell portion, optionally wherein the weight to weight ratio of the phospholipid conjugated to polyethylene glycol to the combined weight of the cationic lipid and hydrophobic polymeric matrix material is from 1 :2 to 1 : 100, such as from 1 :5 to 1 :50;
  • the composition may further comprise a targeting moiety distributed in an outer region of the shell portion, optionally wherein: the targeting moiety is hyaluronic acid and/or folic acid; and/or the targeting moiety is hyaluronic acid and/or folic acid conjugated to a polyethylene portion of a phospholipid conjugated to polyethylene glycol;
  • the average particle size of the lipid-polymer nanoparticles may be from 50 to 500 nm (e.g. the average particle size of the lipid-polymer nanoparticles may be from 75 to 300 nm, such as from 90 to 250 nm, such as from 100 to 135 nm);
  • the composition may have a polydispersity index of from 0.1 to 0.3, such as from 0.1 to 0.29, such as from 0.2 to 0.24;
  • the nanoparticles may have a zeta potential of from +0.1 to +25 mV;
  • the cationic lipid may be selected from salts of one or more of the group consisting of
  • DOTAP 1,2-Dioleoyloxy-3-(trimethylammonium)propane
  • DOGS 1,2-dioleoyl-sn-glycero-3-[(N-(5- amino-1-carboxypentyl)iminodiacetic acid)succinyl]
  • DDAB dimethyldioctadecylammonium
  • the lipid may further comprise a neutral lipid, optionally wherein: the neutral lipid may be selected from one or more of the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and/or the weight to weight ratio of the neutral lipid to the cationic lipid may be from 1 :1 to 1 : 100 w/w;
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • DOPC ,2-dioleoyl-sn-glycero-3-phosphocholine
  • the surfactant may be an amphoteric and/or non-ionic surfactant, optionally wherein the surfactant may be selected from one or more of the group consisting of ethoxylated linear alcohols, ethoxylated alkyl phenols, fatty acid esters, fatty alcohol polyglycosides, alkylpolyglucosides ethyleneoxide/propyleneoxide copolymers, polyalcohols, ethoxylated polyalcohols, mercaptans, amides, alkylpolyglucosides, sultaines, betaines, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelins and lecithins (e.g. the surfactant may be polyvinyl alcohol and/or lecithin);
  • the phospholipid conjugated to polyethylene glycol may be a DSPE-PEG, such as DSPE-PEG2000 or DSPE-PEG5000.
  • the composition may further comprise an active agent, optionally wherein the active agent is distributed in the core portion and/or in the shell portion.
  • the combined weight of the hydrophobic polymeric matrix material and the lipid may be from 40 to 98.9 wt%;
  • the weight of the surfactant may be from 0.1 to 10 wt%
  • the weight of the active agent may be from 1 to 50 wt%, based on the total weight of the composition
  • composition when the composition further comprises a phospholipid conjugated to polyethylene glycol distributed in the shell portion, said phospholipid conjugated to polyethylene glycol may be present in an amount of from 0.1 to 5 wt% of the total weight of the composition;
  • the weightweight ratio of the cationic lipid to hydrophobic polymeric matrix material may be from 1 :3 to 1 :40.
  • said active agent may be selected from one or more of the group consisting of an antibiotic, an anticancer agent and a nutritional agent.
  • antibiotics include, but are not limited to, one or more of the list: aminoglycosides (e.g. gentamycin, kanamycin, streptomycin), beta-lactams (e.g. penicillins, amoxicillin), glycopeptides (e.g. vancomycin), rifamycin (e.g. rifampicin), D-cycloserine, diaryiquino!ines (e.g. bedaquiiine), quinolones (e.g.
  • ciprofloxacin levofloxacin
  • lipopeptides e.g. daptomycin
  • tetracyclines e.g. tetracyclines
  • teixobactin e.g. sulfonamides
  • microlides e.g. ansamycins, streptogramins, chloramphenicol, fusidic acid and monobactams.
  • anti-cancer agents examples include, but are not limited to, one or more of the list: doxorubicin, bleomycin, busulfan, capecitabine, cladribine, decarbazine, docetaxel, vincristine, cisplatin, temsirolimus, etoposide, epiroxaliplatin, mitoxantrone, leucovorin, pemetrexed, pentostain, procarbazine, raltitrexed, streptozocin, fluorouracil, gemcitabine and mitomycin.
  • nutritional agents examples include, but are not limited to, one or more of the list: hexoses (e.g. glucose), pentoses (e.g. fructose), amino acids (e.g. arginine, alanine), and salts (e.g. sodium chloride, potassium chloride, disodium hydrogen phosphate).
  • composition according to the first aspect of the invention and any technically sensible combination of its embodiments, in medicine, provided that the composition further comprises an active agent.
  • a method of treating cancer comprising administering a pharmaceutically effective amount of a composition according to the first aspect of the invention and any technically sensible combination of its embodiments, to a patient in need thereof, wherein the composition further comprises an anti-cancer agent.
  • a composition according to the first aspect of the invention and any technically sensible combination of its embodiments in the manufacture of a medicament for the treatment of cancer, wherein the composition further comprises an anti-cancer agent.
  • composition according to the first aspect of the invention and any technically sensible combination of its embodiments, for use in the treatment of cancer, wherein the composition further comprises an anti-cancer agent.
  • the cancer may be breast cancer, optionally wherein the composition comprises paclitaxel and may optionally further comprise epigallocatechin gallate.
  • a method of treating a microbial infection comprising administering a pharmaceutically effective amount of a composition according to the first aspect of the invention and any technically sensible combination of its embodiments, to a patient in need thereof, wherein the composition further comprises an antibiotic.
  • compositions according to the first aspect of the invention and any technically sensible combination of its embodiments, for use in the treatment of a microbial infection wherein the composition further comprises an antibiotic.
  • a ninth aspect of the invention there is provided a method of manufacturing a lipid-polymer nanoparticle composition for drug delivery as described in the first aspect of the invention and any technically sensible combination of its embodiments, the method comprising the steps of:
  • the power of the ultrasonication is from 20 to 70 W, such as 50 W;
  • the concentration of the hydrophobic polymeric matrix material in the organic solvent is from 0.1 mg/ml_ to 50 mg/ml_, such as from 1 mg/ml_ to 25 mg/ml_, such as from 5 to 15 mg/ml_;
  • the concentration of the lipid in the organic solvent is from 2x1 O 3 mg/ml_ to 15 mg/ml_, such as from 0.02 mg/ml_ to 7.5 mg/ml_, such as from 0.1 to 4.5 mg/ml_;
  • the concentration of the surfactant in the aqueous solution is from 0.05 to 5% w/v, such as from 0.1 to 1 % w/v;
  • the period of time for the sonication after complete addition of the polymer solution is from 1 to 20 seconds, such as from 5 to 10 seconds;
  • the dropwise addition in step (c) above may use a flow rate of from 0.0167 mL/sec to 1 mL/sec to form the nanoparticles, for example the flow rate of the addition of polymer to the aqueous solution may be from 0.05 mL/sec to 1 mL/sec, such as from 0.1 mL/sec to 0.2 mL/sec;
  • the organic solvent is selected from one or more of the group consisting of acetone, dichloromethane, chloroform, toluene, pentane, hexane, heptane, octane, nonane, n-decane, n-dodecane, benzyl chloride, hexadecane, diethyl ether, ethyl acetate, cyclohexane, chloromethane, trichloroethylene (TCE), benzene, bromodichloromethane, vinyl chloride, t
  • TCE
  • the volume to volume ratio of the organic solvent to the aqueous solution is from 1 : 1 to 1 :50, such as from 1 : 5 to 1 :25, such as 1 :20;
  • the polymer solution further comprises an active agent, optionally wherein the concentration of the active agent in the organic solvent is from 0.1 to 25 mg/ml_, optionally wherein the polymer solution further comprises water and the polymer solution is formed by adding an aqueous solution of the active agent to a solution comprising an organic solvent, a hydrophobic polymeric matrix material and a lipid;
  • the aqueous solution further comprises a phospholipid conjugated to polyethylene glycol, optionally wherein: the concentration of the phospholipid conjugated to polyethylene glycol in the aqueous solution is from 0.1 to 5% w/v; and/or the phospholipid conjugated to polyethylene glycol further comprises a targeting molecule that is conjugated to the polyethylene glycol portion of the phospholipid conjugated to polyethylene glycol (e.g. the targeting moiety may be hyaluronic acid and/or folic acid);
  • Figure 1 Physicochemical properties of PLGA nanoparticles and PLGA/DOTAP nanoparticles, (a), (b) SEM images of PLGA nanoparticles and PLGA/DOTAP nanoparticles (c), (d) Zeta potential of PLGA nanoparticles and PLGA/DOTAP nanoparticles (e), (f) Dynamic Laser Scattering of PLGA nanoparticles and PLGA/DOTAP nanoparticles.
  • Lipid coating i.e. DOTAP
  • DOTAP DOTAP
  • the bacterial culture was incubated with propidium iodide (PI) in the presence of blank nanoparticles (i) or with Pl- encapsulated nanoparticles (ii), together with Syto9, for 4 h at 37 °C prior to imaging by confocal laser scanning microscope (CLSM).
  • CLSM confocal laser scanning microscope
  • the top, middle and bottom panels represent the images of PI, Syto9 and the PI+Syto9 overlay, respectively.
  • Scale bar 20 urn (a-b) and 5 urn (c).
  • PLGA/DOTAP nanoparticles can bind efficiently to both Gram-positive and Gram-negative bacteria
  • CLSM Confocal laser scanning microscopy
  • Gram positive bacteria S. thermophilus LMD-9 (i), E. faecalis OG1 RF (ii), S. aureus ATCC25923 (iii) and S. aureus USA300 (iv), and Gram-negative bacteria E. coli BL21 (v), P. stewartii R067d (vi) and P. aeruginosa PA01 (vii), after co-staining with propidium iodide (Pl)- encapsulated PLGA/DOTAP nanoparticles and Syto9 for 4 h at 37 °C.
  • Pl propidium iodide
  • the top, middle and bottom panels represent the images of the PI, Syto9 and the PI+Syto9 overlay, respectively
  • PLGA/DOTAP nanoparticles can bind efficiently to bacterial biofilms.
  • An eight-hour biofilm culture was incubated with Pl-encapsulated nanoparticles and Syto9 for 4 h at 37 °C prior to imaging by CLSM.
  • the top, middle and bottom panels represent the images of the PI, Syto9 and the PI+Syto9 overlay, respectively. Scale bar: 20 urn.
  • PLGA/DOTAP nanoparticles prolong the growth inhibitory effect of antibiotics on bacterial biofilms.
  • a 48 h E. coli UTI89 pre-formed biofilm was subjected to ampicillin (a) or D-cycloserine (b) treatment for continuous 16 h (0+16) or treatment for 1 h followed by 15 h (1+15) of recovery on non-selective medium at 37 °C.
  • the antibiotics were either presented in free-form or encapsulated in LPN (i.e. PLGA/DOTAP nanoparticles).
  • the biofilm viability was determined based on the biofilm metabolic activity in terms of ATP content.
  • FIG 8. PLGA and PLGA/DOTAP nanoparticles uptake by non-phagocytic cells, including mouse fibroblasts 3T3 (a-b), human keratinocytes HaCaT (c-d) and human breast cancer cells MCF-7 (e-f).
  • Cellular uptake of PLGA (a, c and e) and PLGA/DOTAP (b, d and f) nanoparticles by different mammalian cell lines was evaluated.
  • the cell culture was incubated with propidium iodide (Pl)-encapsulated nanoparticles, together with Syto9 and/or DAPI for 4 h at 37 °C, 5% C0 2 prior to imaging by confocal scanning laser microscope (a-d).
  • Pl propidium iodide
  • DAPI and PI stains are represented by the green, blue and red, respectively.
  • e-f the cell culture was incubated with Nile red-encapsulated nanoparticles, together with DAPI for 4 h at 37 °C, 5% CO2 prior to imaging. Scale bar: 50 pm (i) and 10 pm (ii, e and f).
  • Figure 9 PLGA and PLGA/DOTAP nanoparticles are non-toxic. Cytotoxicity of blank PLGA nanoparticles and PLGA/DOTAP nanoparticles on (A) MCF-7 cells (human breast cancer cells) and (B) HEK (Human Epidermal Keratinocytes) by exposing the cells to different concentrations of nanoparticles for 24 h at 37 °C, 5% CO2. The cell viability was assessed using the CCK-8 bioassay and normalized to the untreated controls.
  • FIG. 10 The clearance of intracellular S. aureus USA300 from the host keratinocyte by free or LPN-encapsulated antibiotics.
  • the S. aureus USA300-infected keratinocytes were treated by Van (vancomycin @ 250 pg/mL) and/or Gen (gentamicin @ 250 pg/mL) in free formulation or LPN formulation (i.e. PLGA/DOTAP) for 20 h at 37 °C, 5% CO2 .
  • PLGA/DOTAP nanoparticles increase the survival rate of protozoa, T. pyriformis in a bacterial co-culture by enhancing the therapeutic effect of antibiotics.
  • T. pyriformis (10000 cells/mL) was co-cultured with E. faecalis OG1 RF (1x10 L 8 cells/mL) for 8 h prior to the antibiotic treatment (i.e. free ampicillin or ampicillin-encapsulated LPN @ final ampicillin concentrations of 0, 5, 50 or 250 pg/mL) (a).
  • the survival rate of T. pyriformis was determined by microscopic counting 20 h and 40 h post-antibiotic treatments, and normalized by the initial T. pyhformis count (b). The corresponding E.
  • faecalis OG1 RF concentration at 20 h and 40 h were determined by the drop plate method (c). Sidak’s corrections for multiple comparisons were performed and significant differences are indicated as follows: * P ⁇ 0.05, ** P ⁇ 0.01 and *** P ⁇ 0.001. ns. Not significant.
  • FIG. 12 PLGA/DOTAP nanoparticles mediate intracellular antibiotic delivery.
  • T. pyhformis (10000 cells/ml) was co-cultured with E. faecalis OG1 RF (1c10 L 8 cells/ml) for 8 h prior to the antibiotic treatment either by the free-form ampicillin (a) or ampicillin-encapsulated LPN (b) at final ampicillin concentrations of 0 (i), 5 (ii), 50 (iii) or 250 (iv) pg/mL.
  • the protozoa- bacteria co-culture was stained by Syto9 and propidium iodide (PI), and viewed under a confocal laser scanning microscope.
  • PI propidium iodide
  • Images (c) and (d) are magnifications of images (a-iv) and (b-iv), respectively.
  • the top, middle and bottom panels represent the images of the Syto9, PI and the PI+Syto9 overlay, respectively.
  • Scale bar 50 urn (a-b) and 20 urn (c-d).
  • FIG. 17 In vivo antitumor efficacy in a subcutaneous xenograft model of human breast cancer (MCF-7) cells. Tumor growth of MCF-7 tumor-bearing mice that received intravenous injection of various formulations at an equivalent dose to paclitaxel 5 mg/kg and EGCG 2.5 mg/kg. The red arrows indicate the time points of injections (days 0, 7, and 14).
  • Figure 18 depicts in cartoon format a formulated LPN according to the current invention.
  • a PLGA core portion (130) comprising active molecules (140) is surrounded by a shell portion comprising of a cationic lipid DOTAP (1 10), and a phospholipid conjugated to polyethylene glycol DSPE-PEG (120).
  • This invention relates to a lipid-polymer hybrid nanoparticles (LPN) system for sustained release of drugs, such as antimicrobials or anticancer drugs, and methods of using and manufacturing the same.
  • the current invention relates to the preparation and use of LPN systems that are designed to entrap (e.g. one or more) drugs at high drug loading efficiencies, and with sustained and controlled releasing capabilities.
  • the disclosed delivery system can enhance the therapeutic efficacy and efficiency, as well as reduce the dosage frequency.
  • Examples disclosed below show that the LPN delivery system can be used to release hydrophobic, amphiphilic or hydrophilic antibiotics in a sustained manner, and with high affinity to both Gram-positive and Gram-negative bacteria, growing either as planktonic cells or biofilms or as pathogens residing in mammalian cells.
  • the encapsulation of anticancer drugs, and efficacy of these anticancer-LPN will also be demonstrated.
  • lipid-polymer nanoparticle composition for drug delivery comprising: a core portion comprising a hydrophobic polymeric matrix material; and
  • a shell portion comprising a lipid and a surfactant
  • the lipid comprises a cationic lipid
  • the weighhweight ratio of the cationic lipid to hydrophobic polymeric matrix material is from 1 :1 to 1 :50.
  • compositions disclosed herein may have any suitable particle size, provided that the particles can still be described as a nanoparticle (i.e. less than 1000 nm in diameter).
  • the LPNs disclosed herein may have an average particle size of from 50 to 500 nm, such as from 75 to 300 nm, such as from 90 to 250 nm, such as from 100 to 135 nm.
  • the nanoparticulate size may help the LPNs penetrate to their desired sites of action. Details of how to measure the average particle size are provided in the experimental section below.
  • compositions disclosed herein may have any suitable polydispersity index. However, a polydispersity index of from 0.1 to 0.3, such as from 0.1 to 0.29, such as from 0.2 to 0.24 may be used in certain embodiments. Details of how to measure the polydispersity index are provided in the experimental section below.
  • the compositions disclosed herein will generally have a positive zeta potential, due to the presence of the cationic lipid in the outer shell. Any suitable positive zeta potential may be used in composition described herein.
  • nanoparticles disclosed herein may have a zeta potential of from +0.1 to +25 mV. Details of how to measure the zeta potential are provided in the experimental section below.
  • the disclosed LPN delivery system may be provided as a blank or with one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, such as from 1 to 4) active agents encapsulated within the capsule. If an LPN system described herein is provided as a blank (i.e. with no active agent encapsulated), one or more active agents may be encapsulated using the techniques discussed below to achieve the introduction of such materials to the blank LPN. Advantages associated with the LPN delivery system disclosed herein include controlled and sustained release of active agents (drugs), the ability to encapsulate multiple active agents, low toxicity to mammalian cells, high binding affinity to bacterial cell membrane/biofilm matrix, the ability to be targeted at and enter cancer cells and the like.
  • active agents drug
  • composition described herein may enable each of these agents to be released at a suitable rate to obtain the desired overall concentration steady state window in a subject for each drug.
  • the encapsulation efficiency of the active agents may be enhanced relative to other delivery systems, the LPNs may be produced in a one-step process and this process also enables the encapsulation of multiple drugs.
  • the word“comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features.
  • the word“comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word“comprising” may be replaced by the phrases“consists of” or“consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention.
  • the word“comprising” and synonyms thereof may be replaced by the phrase“consisting of” or the phrase“consists essentially of’ or synonyms thereof and vice versa.
  • the term“lipid-polymer nanoparticle” relates to a material having a core portion formed by a polymeric matrix material and a shell portion that contains at least a lipid and a surfactant.
  • an active agent may be distributed in the core portion and/or in the shell portion, depending on the nature of the active agent in question. For example, if the active agent is hydrophobic, then the active agent will be substantially (i.e. >90 wt%, such as >95 wt%, such as >99 wt%, such as >99.999wt%) distributed within the core portion of the composition. If the active agent is hydrophilic, then the active agent will be substantially (i.e.
  • the distribution of the active agent will be influenced by the polar properties of compound in question and certain active agents may be distributed in both the core and shell portions of the composition.
  • hydrophilic is generally understood to describe a substance that has a high affinity for water.
  • a hydrophilic material may be one that is able to be dissolved in, be mixed with, be wetted by or absorbs water.
  • hydrophilic polymer has a high affinity for aqueous solutions.
  • hydrophobic is generally understood to describe a substance that repels water.
  • a hydrophobic material may include materials that do not dissolve in, be mixed with, be wetted by water or absorb an appreciable amount of water.
  • hydrophobic polymer refers to a polymer having a low affinity for aqueous solutions.
  • amphiphilic when used herein refers to a material that displays both hydrophilic and hydrophobic properties. Typically such materials must have at least two regions - one that is hydrophilic and one that is hydrophobic, but may have more than one region of each type.
  • Typical amphiphilic compounds include materials such as fatty acids and lipoproteins, as well as copolymers (i.e. block copolymers) having blocks that carry hydrophilic and hydrophobic groups.
  • active agent when used herein may refer to a substance useful for the treatment of or the prevention of a condition affecting a human or other animal. Said condition may be a disease, a disorder or a physiological condition. It will be appreciated that the active agent may not directly affect the underlying condition, but may be used as an adjuvant with a further active agent to enhance the effectiveness of the other active agent. Thus, the term“active agent” herein incudes all classes of active agents, whether adjuvant or therapeutic, that may be provided to a subject through oral administration. In certain embodiments, the term“active agent” may also be used herein with reference to nutraceuticals, cosmeceuticals and a nutritional agent, as discussed in more detail below.
  • hydrophobic polymer refers to a polymer having a low affinity for aqueous solutions including water.
  • hydrophobic polymers may include polymers that do not dissolve in, be mixed with, or be wetted by water.
  • hydrophobic polymers may also include polymers that do not absorb an appreciable amount of water.
  • the hydrophobic polymeric matrix material used in the present invention may be a natural polymer or a synthetic polymer.
  • the term "natural polymer” as used herein refers generally to a polymeric material that may be found in nature. Examples of a natural hydrophobic polymer include, but are not limited to, natural rubber and alkylated celluloses, such as ethyl cellulose.
  • synthetic hydrophobic polymers include, but are not limited to, polyolefin, polystyrene, polyester, polyamide, polyether, polysulfone, polycarbonate, polyurea, polyurethane, polysiloxane, copolymers thereof, and blends thereof.
  • the hydrophobic polymer is preferably biocompatible. That is, the hydrophobic polymer is preferably a material that does not cause adverse side-effects in a subject following administration (e.g. capable of interacting with a biological system without causing cytotoxicity, undesired protein or nucleic acid modification or activation of an undesired immune response) and, more preferably, it is a material that can be degraded in vivo (e.g. in hours, days, months or years). Disintegration may for instance occur via hydrolysis, may be catalyzed by an enzyme and may be assisted by conditions to which the microparticles are exposed to in vivo.
  • hydrophobic polymers include, but are not limited to, oligomers of glycolide, lactide, polylactic acid, polyesters of a-hydroxy acids, including lactic acid and glycolic acid, such as the poly(a-hydroxy) acids including polyglycolic acid, poly(D,L-lactic-co-glycolic acid) (PLGA), poly-L-lactic acid (PLLA), and terpolymers of D,L-lactide and glycolide; e-caprolactone and e-caprolactone copolymerized with polyesters; polylactones and polycaprolactones including poly(caprolactone) (PCL), poly(s-caprolactone), poly(valerolactone) and poly(gamma-butyrolactone); polyanhydrides; polyorthoesters; polydioxanone; and other biologically degradable polymers that are nontoxic or are present as metabolites in the body.
  • poly(a-hydroxy) acids including polyglycolic acid, poly
  • hydrophobic polymers include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, nitro celluloses, polymers of acrylic and methacrylic esters, ethyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(laurylmeth
  • hydrophobic polymer or a multiple (i.e. 2, 3, 4, or 5) hydrophobic polymer blend may be used as the hydrophobic polymeric matrix material to form the core of the LPN.
  • any suitable ratio can be used, depending on the desired properties to be obtained by the core, which may be readily determined by a person skilled in the art of such formulation techniques.
  • the hydrophobic polymeric matrix material may be selected from one or more of the group consisting of poly(glycolic-co-L,D-lactic acid) (PGLA), poly(L-lactide) (PLLA), poly(£-caprolactone) (PCL), poly(glycolide) (PGA), poly(lactide) (PLA), alginate, chitosan, polysaccharide, hyaluronic acid, and co-polymers thereof.
  • the hydrophobic polymeric matrix material may be poly(glycolic-co-L,D-lactic acid) (PGLA).
  • PGLA poly(glycolic-co-L,D-lactic acid)
  • PLGA poly(L,D-lactic-co-glycolic acid)
  • the shell portion may comprise a lipid and a surfactant.
  • the lipid forms a lipid shell around the core portion, and lipid shell further comprises the surfactant.
  • the surfactant has a hydrophilic head and a hydrophobic tail, with the head positioned on the outer surface of the shell portion and the tail surrounded by the lipid of the lipid shell.
  • the lipid used to form the lipid shell must contain a cationic lipid.
  • Any suitable cationic lipid may be used to form a composition according to the current invention.
  • suitable cationic lipids that may be used in compositions described herein include, but are not limited to, salts of one or more of the group consisting of 1 ,2-Dioleoyloxy-3- (trimethylammonium)propane (DOTAP), 1 ,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1- carboxypentyl)iminodiacetic acid)succinyl] (DOGS), and dimethyldioctadecyl-ammonium (DDAB), optionally wherein the cationic lipid is a salt of DOTAP.
  • DOTAP 1,2-Dioleoyloxy-3- (trimethylammonium)propane
  • DOGS 1,2-dioleoyl-sn-glycer
  • a cationic lipid requires a suitable anionic counterion, which may be any suitable counterion.
  • suitable counterions include, but are not limited to chloride, bromide, and fluoride.
  • the cationic lipid may be present relative to the hydrophobic polymeric matrix material in a weight to weight ratio of from 1 : 1 to 1 :50 (e.g. the weightweight ratio of the cationic lipid to hydrophobic polymeric matrix material may be from less than 1 : 1 (e.g. 1 : 1.1 or 1 :2) to 1 :50, such as from 1 :3 to 1 :40).
  • the surfactant may be an ionic or non-ionic surfactant and combinations thereof.
  • Ionic surfactants include cationic surfactants, anionic surfactants and amphoteric surfactants, all of which may be used in the current invention.
  • the surfactant is an amphoteric and/or non-ionic surfactant.
  • the surfactant may be a phospholipid.
  • Suitable surfactants may be selected from one or more of the group consisting of ethoxylated linear alcohols, ethoxylated alkyl phenols, fatty acid esters, fatty alcohol polyglycosides, alkylpolyglucosides ethyleneoxide/propyleneoxide copolymers, polyalcohols, ethoxylated polyalcohols, mercaptans, amides, alkylpolyglucosides, sultaines, betaines, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelins and lecithins.
  • the surfactant may be polyvinyl alcohol and/or lecithin.
  • the use of phospholipids as the whole or part of the surfactant may be beneficial if the particles are to be targeted towards bacteria, because they can have a strong interaction with the cell membrane of bacteria, as well as an antibiotic effect.
  • a neutral lipid may be added to the composition, where it forms part of the lipid shell.
  • Any suitable neutral lipid may be used to form part of the lipid shell.
  • suitable neutral lipids include, but are not limited to 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
  • DOPE 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • Any suitable amount of the neutral lipid may be used in the composition, provided that it is provided in a weight to weight ratio of at most 1 : 1 with respect to the cationic lipid.
  • An example of a suitable amount of neutral lipid in the LPN composition may be provided by a neutral lipid present in an amount of from 1 : 1 to 1 : 100 relative to the weight of the cationic lipid in the composition (i.e. weight to weight ratio of the neutral to cationic lipid may be from 1 : 1 to 1 : 100 w/w).
  • the composition may further comprise a phospholipid conjugated to polyethylene glycol.
  • a phospholipid conjugated to polyethylene glycol As will be appreciated, the hydrophobic tail of the phospholipid will form part of the lipid layer and points towards the core of the composition, with the hydrophilic head (i.e. the phosphate, ammonium and polyethylene glycol) being found in the outermost part of the shell portion.
  • Any suitable phospholipid conjugated to polyethylene glycol may be used in the compositions disclosed herein. Examples of suitable materials include, but are not limited to a DSPE-PEG, such as DSPE-PEG2000 or DSPE-PEG5000.
  • the amount of phospholipid-conjugated polyethylene glycol present in the composition may be from 1 :2 to 1 : 100, such as from 1 : 5 to 1 :50relative to the weight of the combined weight of the hydrophobic polymeric matrix material and cationic lipid (i.e. the weight to weight ratio of the phospholipid-conjugated polyethylene glycol to the combined weight of the hydrophobic polymeric matrix material and cationic lipid is from 1 :2 to 1 : 100, such as from 1 :5 to 1 :50).
  • the introduction of these conjugates may help to improve the stability of the LPNs by preventing aggregation of the LPNs.
  • a targeting moiety onto an outer region of the shell portion. This may allow the compositions described herein to target and enter into specific cells in a subject. Any suitable targeting moiety may be used in the compositions described herein. Examples of suitable targeting moieties include, but are not limited to hyaluronic acid and/or folic acid. In order to function, these targeting moieties need to be presented on the outer surface of the shell portion. This may be achieved by conjugating (i.e.
  • hyaluronic acid and/or folic acid or other targeting moiety
  • a hydrophilic portion of a surfactant or, more particularly, to a polyethylene portion of a phospholipid conjugated to polyethylene glycol, which may be achieved as discussed below.
  • the targeting moiety is conjugated to a polyethylene portion of a phospholipid conjugated to polyethylene glycol the molecule may help to enhance the stability of the LPN as discussed above.
  • the combined weight of the hydrophobic polymeric matrix material and the lipid may be from 40 to 98.9 wt%, the weight of the surfactant may be from 0.1 to 10 wt%, and the weight of the active agent may be from 1 to 50 wt%, based on the total weight of the composition.
  • the composition further comprises a phospholipid conjugated to polyethylene glycol distributed in the shell portion, said phospholipid conjugated to polyethylene glycol may be present in an amount of from 0.1 to 5 wt% of the total weight of the composition.
  • Oral administration is still considered the preferred route for administrating therapeutic agents because of its low cost, ease of administration and high level of patient compliance.
  • compositions disclosed herein may be suited to encapsulating any suitable active agent, such as one or more of an antibiotic, an anticancer agent and a nutritional agent.
  • suitable active agent such as one or more of an antibiotic, an anticancer agent and a nutritional agent.
  • the compositions disclosed herein may be suitable for use in medicine. As such, there is disclosed a use of a composition as described herein in medicine, when the composition contains an active agent.
  • composition may be particularly suited to the treatment of cancer.
  • the invention also relates to:
  • AA a method of treating cancer, comprising administering a pharmaceutically effective amount of a composition as described above to a patient in need thereof, wherein the composition comprises an anti-cancer agent (e.g. comprises an anti-cancer agent and a nutritional agent);
  • an anti-cancer agent e.g. comprises an anti-cancer agent and a nutritional agent
  • composition as described herein in the manufacture of a medicament for the treatment of cancer, wherein the composition comprises an anti-cancer agent (e.g. comprises an anti-cancer agent and a nutritional agent); and
  • an anti-cancer agent e.g. comprises an anti-cancer agent and a nutritional agent
  • composition as described herein for use in the treatment of cancer, wherein the composition comprises an anti-cancer agent (e.g. comprises an anti-cancer agent and a nutritional agent).
  • an anti-cancer agent e.g. comprises an anti-cancer agent and a nutritional agent.
  • any suitable anti-cancer agent may be used in the compositions for use in the treatment of cancer.
  • suitable anti-cancer agents include, but are not limited to, one or more of the list: doxorubicin, bleomycin, busulfan, capecitabine, cladribine, decarbazine, docetaxel, vincristine, cisplatin, temsirolimus, etoposide, epiroxaliplatin, mitoxantrone, leucovorin, pemetrexed, pentostain, procarbazine, raltitrexed, streptozocin, fluorouracil, gemcitabine, and mitomycin.
  • compositions for use in the treatment of cancer may also include a nutritional agent.
  • “Nutritional agent” refers to any material that may be beneficial to the human or animal body that may be obtained from a source of food. Such materials may include, but are not limited to, carbon metabolites (e.g. glucose, fructose, fumarate, etc.), electron acceptors (e.g.
  • the composition may comprise paclitaxel and optionally further comprises epigallocatechin gallate. Said compositions may be particularly suited to the treatment of breast cancer.
  • cancers include, but are not limited to, a hyperproliferative vascular disease (such as intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion), a hyperproliferative skin disease (such as psoriasis) and cancer (such as adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain tumours, CNS tumours, breast cancer, Castleman disease, cervical cancer, colon cancer, rectum cancer, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal cancer, hypopharyngeal cancer, leukemia (e.g.
  • a hyperproliferative vascular disease such as intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion
  • acute lymphocytic acute myeloid, chronic lymphocytic, chronic myeloid, chronic myelomonocytic
  • liver cancer e.g. small cell or non small cell
  • lung cancer e.g. small cell or non small cell
  • lung carcinoid tumour e.g. lymphoma
  • composition may also be particularly suited to the treatment of a microbial infection.
  • invention also relates to:
  • composition as described above to a patient in need thereof, wherein the composition comprises an antibiotic (e.g. comprises an antibiotic and a nutritional agent);
  • an antibiotic e.g. comprises an antibiotic and a nutritional agent
  • composition as described herein in the manufacture of a medicament for the treatment of a microbial infection, wherein the composition comprises an antibiotic (e.g. comprises an antibiotic and a nutritional agent); and
  • an antibiotic e.g. comprises an antibiotic and a nutritional agent
  • BC a composition as described herein for use in the treatment of a microbial infection, wherein the composition comprises an antibiotic (e.g. comprises an antibiotic and a nutritional agent).
  • an antibiotic e.g. comprises an antibiotic and a nutritional agent.
  • any suitable antibiotic may be used in the compositions for use in the treatment of microbial infections.
  • suitable antibiotics include, but are not limited to, one or more of the list: aminoglycosides (e.g. gentamycin, kanamycin, streptomycin), beta-lactams (e.g. penicillins, amoxicillin), glycopeptides (e.g. vancomycin), rifamycin (e.g. rifampicin), D- cycloserine, diarylquinoiines (e.g. bedaquiline), quinolones (e.g. ciprofloxacin, levofloxacin), lipopeptides (e.g. daptomycin), tetracyclines, teixobactin, sulfonamides, microlides, ansamycins, streptogramins, chloramphenicol, fusidic acid and monobactams.
  • aminoglycosides e.g. gent
  • compositions for use in the treatment of a microbial infection may also include a nutritional agent.
  • “Nutritional agent” refers to any material that may be beneficial to the human or animal body that may be obtained from a source of food and suitable nutritional agents are mentioned above.
  • Specific materials that may be mentioned herein in combination with antibiotics include carbon metabolites (e.g. glucose, fructose, fumarate, etc.), electron acceptors (e.g. nitrate, peroxide, etc.).
  • carbon metabolites e.g. glucose, fructose, fumarate, etc.
  • electron acceptors e.g. nitrate, peroxide, etc.
  • microbial infections include, but are not limited to, wound infections caused by Staphylococcus aureus, group A b-hemolytic streptococci (e.g. Streptococcus pyogenes), Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and others, either as monospecies or multispecies infection, urinary tract infections, meningitis, endocarditis, and bacteriaemia.
  • group A b-hemolytic streptococci e.g. Streptococcus pyogenes
  • Enterococcus faecalis Enterococcus faecalis
  • Escherichia coli Escherichia coli
  • Pseudomonas aeruginosa Pseudomonas aeruginosa and others, either as monospecies or multispecies infection
  • urinary tract infections meningitis, endocardi
  • treatment includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.
  • patient and“patients” include references to mammalian (e.g. human) patients.
  • subject or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, came, and, most preferably, a human.
  • the subject is a subject in need of treatment or a subject with a disease or disorder.
  • the subject can be a normal subject.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
  • “effective amount” and“suitable amount” and variants thereof refer to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease).
  • the effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).
  • the LPNs may be provided for oral administration to a subject for effecting treatment of said subject.
  • the formulation may further comprise a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.
  • the LPNs described herein may be packaged in a tablet or, more particularly, a capsule (e.g. a gelatin capsule) for ease of administration, where said tablet/capsule is selected to release the LPNs shortly after delivery to the stomach.
  • any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound in the formulation may be determined routinely by the skilled person.
  • a solid oral composition such as a tablet or capsule may contain from 0.5 to 20% (w/w) active ingredient(s) in the LPNs; from 50 to 99% (w/w) of the LPNs (including the drug(s)), from 0 to 50% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment.
  • each drug (whether hydrophobic or hydrophilic) included in each LPN will depend on the desired eventual dosage of the drug in question.
  • each drug (whether hydrophobic or hydrophilic) loaded into the LPNs may be present in an average amount of from 1 wt% to 50 wt%, such as from 5 wt% to 45 wt%, such as from 10 wt% to 30 wt% (e.g. from 15 wt% to 25 wt%) of the total weight of the LPN.
  • the total weight percentage of all drugs in the LPN may be from 1 to 50 wt%, such as from 5 wt% to 45 wt%, such as from 10 wt% to 30 wt% (e.g. from 15 wt% to 25 wt%).
  • the formulations may be administered at varying therapeutically effective doses to a patient in need thereof.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
  • the dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of each drug.
  • the medical practitioner or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient.
  • the above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • aspects of the invention described herein may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.
  • compositions disclosed herein may also be suitable for the delivery of nutraceuticals, cosmeceuticals and food-based nutrients, whether in the treatment of a chronic condition or otherwise.
  • the terms“drug”, “first drug” and“second drug” may be applied to nutraceuticals, cosmeceuticals and food- based nutrients.
  • the “first drug” will relate to hydrophilic nutraceuticals, cosmeceuticals and food-based nutrients
  • the second drug will relate to hydrophobic nutraceuticals, cosmeceuticals and food-based nutrients.
  • nutraceuticar is a portmanteau of "nutritional” and “pharmaceutical” and refers to foods thought to have a beneficial effect on human health it can also refer to individual chemicals which are present in common foods. Many such nutraceuticals are phytonutrients. Nutraceuticals are sometimes called functional foods. Suitable nutraceuticals that may be mentioned herein may be:
  • hydrophilic nutraceuticals which may be selected from, but not limited to, phenolic compounds (such as, for example, Resveratrol), Quercetin, Rutin, polyphenols (such as, for example, Oiigonoi from lychee fruit), catechins, bioactive polysaccharides (such as, for example, Active Hexose Correlated Compound or AHCC), cofactors (such as, for example, pyrroloquinoline quinone (PQQ)), amino acids (such as, for example, arginine and glutamine), and mixtures thereof; and
  • hydrophobic nutraceuticals which may be selected from, but not limited to, mixed carotenoids, carotenoid esters, Curcuminoids (e.g Curcumin), Poiicosanoi, Silymarin, Baicalein, Quercetin, plant sterols, vitamins (such as, for example, Vitamin E and A), alpha lipoic acid, sesquiterpene lactones (such as, for example, partheno!ides), and mixtures thereof.
  • Cosmetical is also a hybrid term incorporating the concept of improving skin appearance by application of active ingredients that often serve a therapeutic role.
  • Suitable cosmeceuticals may include, but are not limited to a desquamating agent, a moisturizer, a depigmenting or pro-pigmenting agent, an anti-glycation agent, an NQ-syntbase inhibitor, a Soreductase inhibitor, a iysyi and/or prolyl hydroxylase inhibitor, an agent for stimulating the synthesis of dermal or epidermal macromolecules and/or for preventing their degradation, an agent for stimulating the proliferation of fibroblasts and keratinocytes and/or keratinocyte differentiation, a muscle relaxant, a compound for reducing irritation, an antimicrobial agent, a tensioning agent, an anti-pollution agent, a free-radical scavenger and mixtures thereof
  • Food-based nutrient refers to any material that may be beneficial to the human or animal body that may be obtained from a source of food. Such materials may include, but are not limited to, a vitamin such as vitamin A, B1 , B2, B3, B6, B12, D, E, biotin, folate, and panothenate; minerals such as calcium, magnesium, selenium, and zinc; an amino acid such as asparagine, carnitine, glutamine, and serine; an antioxidant selected from coenzyme Q10, glutathione, and cysteine; or a metabolite such as lipoic acid, oleic add, choline, inositol, fructose, glucose, and insulin, and mixtures thereof.
  • a vitamin such as vitamin A, B1 , B2, B3, B6, B12, D, E, biotin, folate, and panothenate
  • minerals such as calcium, magnesium, selenium, and zinc
  • an amino acid such as asparagine, carn
  • compositions and uses described herein may employ materials selected from the classes of standard active pharmaceutical ingredients (i.e. active therapeutic agents and/or adjuvants), nutraceuticals, cosmeceuticals and nutritional agents. Any suitable combination of these classes may be used.
  • the material may solely contain active pharmaceutical ingredients or may contain one or more active pharmaceutical ingredients in combination with a nutritional agent and the like.
  • the compositions may comprise (or contain) active pharmaceutical ingredients only.
  • a formulated LPN 100 is provided in Fig. 18.
  • the active agent 140 is substantially distributed throughout the polymeric core 130, with a cationic lipid 1 10 (e.g. DOTAP) forming the lipid shell, along with a phospholipid conjugated to polyethylene glycol 120 (e.g. DSPE-PEG).
  • a cationic lipid 1 10 e.g. DOTAP
  • polyethylene glycol 120 e.g. DSPE-PEG
  • this general structure may be adapted as needed for the desired active agent and desired site of action (e.g. the phospholipid conjugated to polyethylene glycol may be absent, or it may further comprise a targeting moiety).
  • the LPNs may be formed by a process that enables the rapid and convenient formation of LPNs containing one or more active agents.
  • a method of manufacturing a lipid-polymer nanoparticle composition for drug delivery as described above comprising the steps of:
  • the disclosed fabrication method presents an advanced hybrid nanoparticle that encapsulates and releases different kinds of active agents (e.g. antibiotics), regardless of hydrophobicity, in a controlled manner.
  • This delivery system possesses advantages of both the polymeric-based (i.e. ease of fabrication, high encapsulation efficiency of hydrophobic drugs, sustained and controlled release) and lipid-based nanoparticles (i.e. strong interaction with bacterial cell membrane), while circumventing the disadvantages of these single systems when used alone.
  • this LPN delivery system improves the encapsulation efficiency of all active agents (e.g. antibiotics) used compared to the polymer nanoparticles alone.
  • the present invention can also offer greater versatility in controlling drug release rates by manipulating different particle fabrication parameters, i.e.
  • Cationic lipid can be used to form shell, giving lipid-polymer shell- core particles (i.e. DOTAP, DOGS, DDAB).
  • neutral lipid can be mixed with cationic lipids (i.e. DOPE, DOPC).
  • DOPE cationic lipids
  • Ratio of cationic lipid and polymer This ratio should be kept at or below 1 : 1 w/w (e.g. at or below 1 :2 or at or below 1 :3 as discussed in more detail above) to form the shell/core structure. As noted below in the examples, it is possible to form particles that are useful using a ratio of 1 : 1 or 1 :2 w/w of cationic lipid:polymer, but more consistent particles sizes and particle uniformity may be obtained when the ratio is at or below 1 :3 w/w.
  • Ultra-sonication amplitude and power This can be manipulated to control size of the LPN, which may be set to from 10 to 30% (e.g. 20%) of the maximum amplitude achievable with the ultrasonication probe.
  • the energy applied to the probe is changed from electrical into mechanical energy, which causes a motion that travels through the horn/probe causing the tip to move up and down. The distance of one movement up and down is called its amplitude.
  • the amplitude is adjustable and each probe has a maximum amplitude value (i.e. 100%). For example, a 1.32 cm diameter probe at setting 100% may achieve an amplitude of approximately 120pm, while at setting 50% the amplitude may be approximately 60pm.
  • the use of a lower amplitude may generate an LPN with a smaller particle size (e.g. in the examples below, a 20% setting for amplitude provided particles of the smallest size compared to the other amplitudes tested).
  • Phospholipids i.e. lecithin which are GRAS (Generally Recognized as Safe) grade may be added to the composition because they can have a strong interaction with the cell membrane of bacteria, as well as an antibiotic effect.
  • Other types of surfactants can also be used for preparation of LPN (i.e. PVA). Surfactants are discussed in more detail above and any of said surfactants may be used in the production of LPNs.
  • Phospholipid conjugated PEG i.e. DSPE- PEG2000
  • DSPE- PEG2000 phospholipid conjugated PEG
  • Any suitable concentration of phospholipid conjugated to polyethylene glycol may be used.
  • the concentration of the phospholipid conjugated to polyethylene glycol in the aqueous solution may be from 0.1 to 5% w/v.
  • Concentration of surfactant in aqueous solution It can be manipulated to control size of the LPN. From 0.05 to 5% w/v, such as from 0.1 to 1.0% w/v. For example, the particle size of LPN was smaller when a concentration of from 0.1 to 1 % compared to particles made using a higher concentration (e.g. from 1 to 5% w/v).
  • Sonication power The power (watt) should be kept in the range of 20 to 70 W, such as 50 W or below 50W (e.g. from 20 to 49.9 W). This allows the formation of a W1/O/W2 emulsion to form when the polymer solution includes an active agent (as will be discussed in more detail below). It is believed that the power needs to be kept within the above range or the droplets will be broken and drug encapsulated polymer will be precipitated.
  • Sonication time It can be manipulated to control size of the LPN. It is noted that sonication time referred to herein refers to the time following the complete addition of the polymer solution to the aqueous solution. For example, the period of time for the sonication after complete addition of the polymer solution may be from 1 to 20 seconds, such as from 5 to 10 seconds. It is noted that over 10 seconds, the particle size of the LPNs significantly increased.
  • any suitable concentration of the hydrophobic polymeric matrix material in the organic solvent may be used, provided that an LPN is provided.
  • Suitable concentrations of the hydrophobic polymeric matrix material in the organic solvent include, but are not limited to, from 0.1 mg/mL to 50 mg/mL, such as from 1 mg/mL to 25 mg/mL, such as from 5 to 15 mg/mL.
  • any suitable concentration of the lipid in the organic solvent may be used, provided that the resulting LPN has a positive zeta potential, due to the presence of the cationic lipid.
  • Suitable concentrations include, but are not limited to, from 2x1 O 3 mg/mL to 15 mg/mL, such as from 0.02 mg/mL to 7.5 mg/mL, such as from 0.1 to 4.5 mg/mL.
  • concentrations listed here relate to the entire lipid concentration (i.e. cationic and neutral lipids, but not phospholipids, which are discussed herein separately).
  • step (c) may use any suitable flow rate, for example it may use a flow rate of from 0.0167 mL/sec to 1 mL/sec to form the nanoparticles.
  • the flow rate of the addition of polymer to the aqueous solution may be from 0.05 mL/sec to 1 mL/sec, such as from 0.1 mL/sec to 0.2 mL/sec.
  • the organic solvent may be selected from one or more of the group consisting of acetone, dichloromethane, chloroform, toluene, pentane, hexane, heptane, octane, nonane, n-decane, n-dodecane, benzyl chloride, hexadecane, diethyl ether, ethyl acetate, cyclohexane, chloromethane, trichloroethylene (TCE), benzene, bromodichloromethane, vinyl chloride, trichloroethane, methyl ethyl ketone, methyl isobutyl ketone, methyl tert-butyl ether, vinyl acetate, dichloroethane, chloroethane, trichlorotrifluoroethane, ethylbenzene and isopropylbenzene
  • TCE trichloroethylene
  • organic solvents e.g. acetone
  • the polymeric core may form when the polymer solution is dropped into the aqueous solution.
  • Any suitable amount of organic solvent to aqueous solution may be used.
  • suitable volume to volume ratio of the organic solvent to the aqueous solution include, but are not limited to, from 1 :1 to 1 :50, such as from 1 : 5 to 1 :25, such as 1 :20.
  • the method disclosed above may be particularly suited to the direct encapsulation of an active agent into the resulting LPN.
  • This may be conveniently achieved by adding the active agent to the polymer solution.
  • the active agent may be added to the polymer solution directly or provided as a solution in a suitable solvent (e.g. water, which may be used for a hydrophilic agent), which is added to the polymer solution.
  • a suitable solvent e.g. water, which may be used for a hydrophilic agent
  • an active agent When an active agent is added to the polymer solution it may have a concentration in the organic solvent of from 0.1 to 25 mg/mL.
  • the combined concentration of all active agents may be from 0.1 to 25 mg/mL in the organic solvent.
  • a targeting moiety into the LPN structure.
  • This may be conveniently achieved by the conjugation of a targeting moiety to a phospholipid conjugated to polyethylene glycol.
  • the phospholipid conjugated to polyethylene glycol may be suitably functionalised to allow for the formation of a covalent bond between the polyethylene glycol portion of the conjugate molecule and the targeting moiety.
  • suitable functional groups may include hydroxyl, thiol, amino and the like.
  • the targeting moiety is hyaluronic acid or folic acid
  • the functional group on the polyethylene glycol that may form a covalent bond may be an amino group.
  • the resulting material may be introduced to the method in addition to or in place of a phospholipid conjugated to polyethylene glycol.
  • concentration of the targeting moiety- conjugate in the aqueous solution may be from 0.1 to 5% w/v.
  • the active agent may be a protein or a peptide. While these may be conveniently encapsulated using the method described above where the active agent is introduced during the manufacture of the LPNs, it is also possible to load these compounds into a blank LPN. This may be achieved by adding a solution of a protein or a peptide that is negatively charged to blank, positively-charged LPNs disclosed herein (or vice versa). As will be appreciated, the nanoparticles may be present in a solution or in a dry state. As will be appreciated, the solvents used to form the solutions may be any suitable solvents, such as an aqueous solvent (e.g. water).
  • an aqueous solvent e.g. water
  • LPNs can provide a controllable release profile for one or more active agents. This control may include minimising burst release, followed by the delivery of single or multiple active agents (e.g. antibiotics) to the site of action.
  • active agents e.g. antibiotics
  • the lipid profile of the LPN may, in addition, provide a localized delivery near biofilms or into mammalian cells.
  • PLGA lactide : glycolide molar ratio 50:50, intrinsic viscosity: 1.18 and molecular weight of 45 kDa
  • DOTAP and DSPE-PEG were purchased from Avanti Polar Lipids (Alabaster, AL, USA). All antibiotics, Poly(vinyl alcohol) (PVA) (30,000 - 80,000 MW, 87% hydrolysed) and lecithin (from egg yolk, 399% (TLC), lyophilized powder) were purchased from Sigma-Aldrich. Additional chemicals were obtained commercially at analytical grade.
  • TTB tryptic soy broth
  • TSA agar
  • P. stewartii R067d was cultivated at 30 °C for 24 h while other bacteria were grown at 37 °C for 24 h.
  • the field emission scanning electron microscopy was used to analyze the morphology of LPNs. Briefly, freeze-dried LPNs were uniformly mounted onto metal stubs and sputter coated with gold. Transmission electron microscopy (TEM, JEOL 2100F) was used to observe shell/core structure of PLGA/DOTAP nanoparticles. TEM samples were prepared by the addition of the nanoparticle solution onto a hydrophilic Formvar-coated copper grid for 3 min. Particle size and polydispersity indices were determined by dynamic light scattering (DLS) using HORIBA Nano Particle Analyzer SZ-100. Prior to DLS, the LPNs were suspended in dh ⁇ O and ultrasonicated for 10 s to disperse the LPNs uniformly.
  • DLS dynamic light scattering
  • the zeta potential was measured with the Zetasizer Nano Z (Malvern Co., UK). Data were collected as the average of 20 measurements.
  • the lyophilized nanoparticles were first dispersed in distilled water and sonicated to minimize inter-particle interactions. Examples
  • a hydrophobic polymer and a smaller quantity e.g. from 0.33 - 3.3 mg, such as from 1.0 - 3.3 mg
  • a cationic lipid e.g. 10 mg
  • 10 mg of a hydrophobic polymer and a smaller quantity e.g. from 0.33 - 3.3 mg, such as from 1.0 - 3.3 mg
  • a cationic lipid e.g. 10 mg
  • the active agent(s) or solution of active agent(s)
  • was mixed with the acetone solution to provide a polymer solution such that the active agent is added at 10% w/w relative to the combined weight of the hydrophobic polymer and cationic lipid).
  • the polymer solution was added dropwise into a 20 mL aqueous solution containing a phospholipid conjugated PEG (0.1 to 0.33 mg/mL in 20 mL solution) and 1 % (w/v) of a surfactant under ultra-sonication at 20 % of amplitude, where the sonication power is kept below 50 W.
  • the resulting mixture was sonicated for up to 10 s at the same settings, after which the sonication was stopped. Nanoparticles were formed instantly upon mixing (i.e. contact of the polymer solution with the lipid solution under sonication).
  • the residual acetone in the suspension was evaporated by continuous stirring (250 rpm) of the suspension at room temperature for 4 h.
  • the nanoparticles were washed three times in Amicon tubes (MWCO 100kDa; Millipore) to remove any remaining organic solvent and free antibiotic in distilled water.
  • Polymer nanoparticles were prepared according to General Procedure 1 except that the cationic lipid was not present in the initial acetone solution.
  • Example 1 Preparation of PLGA/DOTAP nanoparticles and encapsulation of various antibiotics compared to PLGA nanoparticles
  • PLGA/DOTAP nanoparticles were prepared using General procedure 1 above, which procedure is a reproducible and scalable self-assembly method.
  • DOTAP (1 ,2-dioleoyl-3- trimethylammonium-propane) was chosen as the shell component to:
  • PLGA poly(lactic-co-glycolic acid)
  • DOTAP 1,2-dioleoyl-3- trimethylammonium-propane
  • 10 mg PLGA poly(lactic-co-glycolic acid)
  • DOTAP 1,2-dioleoyl-3- trimethylammonium-propane
  • a total of 100 pl_ antibiotics (20 mg/ml_) solution was mixed with the acetone solution.
  • the polymer solution was added dropwise into a 20 ml_ aqueous solution containing DSPE-PEG5K (0.1 to 0.33 mg/ml_ in 20 ml_ solution) and 1 % (w/v) of lecithin under ultra-sonication at 20 % of amplitude, where the sonication power is kept below 50 W.
  • the resulting mixture was sonicated for up to 10 s at the same settings, after which the sonication was stopped. Nanoparticles formed instantly upon mixing.
  • the residual acetone in the suspension was evaporated by continuous stirring (250 rpm) of the suspension at room temperature for 4 h.
  • the nanoparticles were washed three times in Amicon tubes (MWCO 100kDa; Millipore) to remove any remaining organic solvent and free antibiotic in distilled water.
  • Table 1 presents the physical properties of PLGA/DOTAP nanoparticles when different weight ratios of PLGA and DOTAP were used in preparing the particles and antibiotics are not added.
  • Table 1 Physical properties of PLGA/DOTAP nanoparticles according to different ratios of DOTAP and PLGA.
  • This LPN system can be prepared with various types of surfactants.
  • lecithin was replaced with PVA
  • the particle size and zeta potential of LPN (1 :3) was 124.4 nm and 25.4 mV, respectively.
  • Different antibiotics with varying degrees of hydrophilicity were encapsulated in PLGA or PLGA/DOTAP nanoparticles using the general procedures 1 and 2 above. Specifically, 10% (w/w) of the specific antibiotic was added. 9 mg of PLGA and 1 mg of DOTAP were dissolved in 1 ml of organic solvent. Then, 100 pi of the antibiotic having a 20 mg/ml concentration was added into the 1 ml solution to provide a polymer solution.
  • the encapsulation efficiency (%) of each antibiotic was determined using HPLC. Table 2 lists the encapsulation efficiencies and the physical properties of LPNs loaded with a single antibiotic.
  • Table 2 Physical properties of PLGA and PLGA/DOTAP nanoparticles loaded with a single antibiotic.
  • our PLGA/DOTAP hybrid nanoparticle comprises a polymer core and a lipid shell
  • the system may possess an ability for encapsulating hydrophobic and hydrophilic molecules in the polymer core and lipid shell, respectively.
  • a hydrophilic antibiotic i.e. ampicillin
  • a hydrophobic antibiotic i.e. rifampicin
  • PLGA/DOTAP nanoparticles containing both vancomycin and gentamycin were also fabricated using general procedure 1 above, where both antibiotics are each added in a single aliquot of 100 mI_ at a concentration of 20 mg/ml_.
  • the physical properties of the resulting nanoparticles are provided in Table 3.
  • Table 3 Physical properties of PLGA and PLGA/DOTAP nanoparticles loaded with two antibiotics.
  • the PLGA/DOTAP nanoparticles exhibited a significantly improved encapsulation efficiency of ampicillin and rifampicin (42.2 ⁇ 7.3% and 83.1 ⁇ 8.9 %) compared to PLGA nanoparticles (26.8 ⁇ 6.1 % and 75.2 ⁇ 7.4 %).
  • Figure 1 shows the scanning electron microscopy (SEM) images, zeta potential and particle size (DLS analysis) of the PLGA nanoparticles and PLGA/DOTAP nanoparticles fabricated.
  • SEM images revealed that PLGA and PLGA/DOTAP nanoparticles were spherical in shape.
  • DLS analysis PLGA/DOTAP nanoparticles were slightly larger than PLGA nanoparticles due to the lipid coating.
  • the negative charge of PLGA i.e. carboxyl terminal end-group
  • the cationic lipid gave PLGA/DOTAP nanoparticles a positive zeta potential, and this is also true for nanoparticles encapsulated with a drug.
  • hydrophilic antibiotics i.e. ampicillin, kanamycin, D-cycloserine
  • PLGA/DOTAP nanoparticles showed more sustained release of these encapsulated hydrophilic antibiotics (see Fig. 2b).
  • Amphiphilic (i.e. amoxicillin) and hydrophobic antibiotics (i.e. rifampicin) also exhibited a more sustained release profile when encapsulated in PLGA/DOTAP nanoparticles. Release was also conducted in cell medium (Tryptone soya broth as shown in Figure 2c), and a similar trend was observed
  • Example 3 PLGA:DOTAP par have affinities with bacteria as compared to PLGA particles with bacteria
  • propidium iodide (Pl)-encapsulated nanoparticles were prepared based on General Procedures 1 and 2 above, where the“active agent” is propidium iodide and 500 mM of propidium iodide was used for loading into a suitable volume of acetone solvent containing 90 mg of PLGA and 10 mg of DOTAP.
  • the resulting nanoparticles were used to stain planktonic bacteria, various other bacteria and bacterial biofilms, as discussed below.
  • the nanoparticles encapsulating PI were incubated with Gram-positive bacteria S. thermophilus LMD-9, E. faecalis OG1 RF, S. aureus ATCC25923, and S. aureus USA300, as well as Gram-negative bacteria E. coli BL21 , P. stewartii R067d and P. aeruginosa PA01 and these results are shown in Figure 5.
  • Pseudomonas aeruginosa PA01 was the only species examined that could not be well labeled by the PLGA/DOTAP nanoparticles. Overall, the use of a lipid coating on a PGLA nanoparticle showed enhanced binding efficiency compared to conventional PLGA nanoparticles to the bacterial cells, regardless of the species ( Figure 5b). The binding affinity of nanoparticles to the bacteria was determined by normalizing the number of bacteria labeled with PI over the total number of bacteria labeled with Syto9.
  • CLSM confocal laser scanning microscope
  • PLGA/DOTAP nanoparticles appeared to bind to E. faecalis OG1 RF and S. aureus USA300 biofilms with higher affinity compared to the PLGA nanoparticles.
  • the enhanced PI intensity was confirmed to be a consequence of accumulated PLGA/DOTAP nanoparticles around the biofilms due to higher binding affinity.
  • Example 4 PLGA/DOTAP inhibit bacteria at lower antibiotic dosage and for a longer period of time
  • each formulation was standardized to an initial antibiotic concentration of 1280 pg/mL.
  • a serial two-fold dilution of the stock antibiotic i.e. , 1280 pg/mL was performed down to an antibiotic concentration of 1.25 pg/mL.
  • Each dilution was further diluted ten times when mixing with the bacterial culture.
  • the effective antibiotic concentration examined was ranging from 128 to 0.125 pg/mL.
  • the effectiveness of the free-form antibiotics were examined in a serial dilution ranging from 128 to 0.125 pg/mL.
  • planktonic cells i.e., E. faecalis OG1 RF, S. aureus USA300 and E. coli UTI89
  • the cultures were added to the antibiotic suspensions to a final bacterial concentration of 8x10 6 cells/ml.
  • Bacteria were exposed to different concentrations of the test material according to Table 4 for 24 h and the bacterial density was determined at optical density 600 nm (OD600).
  • the minimal inhibitory concentration (MIC) was defined as the antibiotic concentration where no visible bacterial growth was observed or the OD600 was less than 5% compared to the untreated control after 24 h of antibiotic exposure.
  • biofilm cultures were pre-established at 37 °C on a 96-well plate or on a PEG-lid prior to exposure to different concentrations of the test material according to Table 4 for 16 h at 37 °C.
  • 200 mI_ of bacterial culture was seeded on a 96-well plate at a final bacterial density of 8 x 10 6 cells mL ⁇ 1 for 8 h (i.e., E. faecalis OG1 RF and S. aureus USA300) while E.
  • coli UTI89 biofilms were established on a PEG-lid for 48 h. The spent liquid medium was discarded and the biofilms was washed gently using TSB to remove the planktonic bacteria. Free or nanoparticle-encapsulated antibiotics prepared in serial concentrations in TSB was added to the biofilms and incubated for 16 h at 37°C. The initial concentration of antibiotics encapsulated in LPNs was determined by HPLC. The viability of bacterial biofilms was assessed based on the ATP content of the biofilms using the BacTiter-Glo microbial cell viability assay (Promega, Singapore), according to the manufacturer’s instructions.
  • the minimal metabolic inhibitory concentration (MMIC) for biofilms was determined based on the ATP content of the biofilms, which is more sensitive for detecting cell viability, and we defined here the MMIC as the antibiotic concentration required to cause 95% or more reduction in ATP content as compared to the untreated control.
  • Table 4 shows the MIC and MMIC values for different bacterial cultures using the encapsulated and the free-form antibiotics. Irrespective of the type of antibiotics and the bacterial species, encapsulating antibiotics in PLGA/DOTAP nanoparticles was generally found to result in at least an 8-fold reduction of the MIC or MMIC compared to the free-from antibiotics. In contrast, when the antibiotics (e.g. ampicillin) were only encapsulated in PLGA nanoparticles, the reduction in the MIC or MMIC was 8-fold or less when compared to free form of the drug.
  • the antibiotics e.g. ampicillin
  • DOTAP was added in a serial concentration (i.e., working concentrations ranging from 640 to 0.625 pg/mL) to the free-form ampicillin at working concentrations ranging from 128 to 0.125 pg/mL.
  • the amount of free-form DOTAP added to each concentration of free-form ampicillin was pre-determined according to the amount of DOTAP present in the PLGA/DOTAP-ampicillin nanoparticle at each dilution.
  • the amount of DOTAP and PLGA present in the PLGA/DOTAP-ampicillin nanoparticle was estimated from the initial free-form DOTAP and PLGA used for nanoparticle fabrication.
  • the blank DOTAP, PLGA nanoparticles and PLGA/DOTAP nanoparticles were found to be non-toxic to all three bacterial species even at the highest concentrations used for antibiotic encapsulation.
  • PLGA/DOTAP nanoparticles can increase the therapeutic effect of antibiotics on both planktonic cells and biofilm inhibition.
  • Table 4 Minimal inhibitory concentration (MIC) and minimum metabolic Inhibitory concentration (MMIC) of free and antibiotics-encapsulated PLGA/DOTAP against different bacterial species.
  • O-Cycioserine (C) >64 64 >64 16 32 >128
  • MIC is defined as the treatment concentration where no visible cell growth can be observed after 24 h of antibiotic exposure.
  • MMIC is defined as 95% or more reduction in biofilm metabolic activity compared to the untreated control after 16 h of antibiotic exposure.
  • UTI89 E. coli UTI89; USA300: S. aureus USA300; OG1 RF: E. faecal is OG1 RF; LPN: Lipid-Polymer hybrid nanoparticles (i.e. PLGA/DOTAP nanoparticles).
  • DOTAP-Ampicillin comprises of free DOTAP mixed with free ampicillin instead of encapsulating ampicillin within DOTAP.
  • serial concentration of the respective carrier was prepared according to the amount of carrier used for ampicillin encapsulation in serial dilutions. In all cases, none of the carriers showed toxicity to all three species even at the highest carrier concentrations.
  • the biofilm viability was determined based on the biofilm metabolic activity indicated by the ATP content. Specifically, the minimal metabolic inhibitory concentration (MMIC) for biofilms was determined based on the ATP content of the biofilms, which is more sensitive for detecting cell viability, and we defined here the MMIC as the antibiotic concentration required to cause 95% or more reduction in ATP content as compared to the untreated control.
  • MMIC minimal metabolic inhibitory concentration
  • encapsulated antibiotic materials in PGLA-DOTAP can become attached to the biofilm on the PEG lid within one hour of treatment.
  • the nanoparticles attached to the biofilm may be able to sustain the almost complete biofilm suppression effect for an extended period of time (due to sustained release of the antibiotic).
  • antibiotic failure in many infectious diseases is often associated with bacterial tolerance and persistence arising as a response to external cues, such as starvation and antibiotic exposure.
  • Metabolic stimulation by carbon supplementation e.g. glucose and fructose
  • antibiotic activity i.e. aminoglycosides
  • both carbon and antibiotics must be co-delivered simultaneously at a specific ratio to the persister cells.
  • the PLGA/DOTAP nanoparticles are an ideal delivery system for both the carbon source and antibiotics. This is demonstrated below by examining the impact of gentamicin and/or fructose, in both free and PLGA/DOTAP formulations, on the survival of S. aureus USA300 persister cells.
  • the persister cells of S. aureus USA300 were harvested from a 20 h planktonic culture (i.e., stationary-phase) grown at 37 °C. The cell culture was washed once and resuspended in phosphate buffer saline (PBS) prior to the desired treatment. The survival of persister cells was determined by the drop plate method, and represented in a color scale with the number of survivals indicated in logarithms of the colony-forming units per ml (log CFU/ml) The detection limit of survival is 2.30 (i.e. 200 cells/ml).
  • gentamicin alone failed to kill the persister cells in either free or PLGA/DOTAP formulation even after 24 h antibiotic exposure (i.e. compared to the untreated control).
  • free fructose was found to decrease the persisters by approximately two logs at 234 pg/mL, and a similar killing effect was achieved by 23.4 pg/mL fructose encapsulated in PLGA/DOTAP nanoparticles, after 24 h exposure. Consistent with previous studies, a synergistic killing effect between fructose and gentamicin was established after treating the persister cells to the compounds for 24 h.
  • the persister cells could be eliminated (i.e. below the detection limit) by both fructose and gentamicin encapsulated in PLGA/DOTAP nanoparticles at a concentration that is at least 10-fold lower than the free formulation (i.e. 23.4 pg/mL fructose and 2 pg/mL gentamicin).
  • PLGA/DOTAP nanoparticles encapsulated with both fructose and gentamicin were prepared according to General Procedure 1 where the active agents are fructose and gentamicin. Specifically, 18 mg of PLGA and 2 mg of DOTAP were dissolved in 1 mL of organic solvent. 5 mg of fructose and 0.5 mg of gentamicine was dissolved in 200 pi of water. This aqueous phase was added into the 1 mL polymer solution. These results suggest that a LPN, such as PLGA/DOTAP nanoparticles, can be used to deliver multiple active ingredients, other than antibiotics, for different therapeutic strategies.
  • Example 6 PLGA/DOTAP mammalian cells
  • the LPNs disclosed herein are also designed for treatment of infections related to intracellular pathogens by transporting antimicrobials into mammalian cells where pathogens reside. This is particularly important for hydrophilic antibiotics since these antimicrobial agents cannot penetrate efficiently across the cell membrane based on simple diffusion. With the DOTAP coating, it is hypothesized that the LPN can interact strongly with the cell membrane, allowing for the internalization of these nanoparticles.
  • PI propidium iodide
  • Nile red-encapsulated nanoparticles were conducted using non-phagocytic mammalian cells, including mouse fibroblasts (3T3), human keratinocytes (HaCaT) and human breast cancer cells (MCF-7).
  • Propidium iodide (Pl)-encapsulated nanoparticles were prepared based on General Procedures 1 and 2 above, where the“active agent” is propidium iodide and 500 mM of propidium iodide was used for loading into a suitable volume of acetone solvent containing a combined weight of 90 mg of PLGA and 10 mg of DOTAP.
  • Nile red-encapsulated nanoparticles were also prepared based on General Procedures 1 and 2 above, where 0.1 mg of Nile red was used for loading into a 9 mg of PLGA and 1 mg of DOTAP.
  • the cell culture was incubated with PI- or Nile red-encapsulated nanoparticles, together with Syto9 and/or DAPI for 4 h at 37 °C, 5% CO2 prior to imaging by confocal scanning laser microscope.
  • the PLGA/DOTAP nanoparticles showed improved intracellular uptake of PI in HaCaT and 3T3 cells or Nile red in MCF-7 cells compared to the PLGA nanoparticles.
  • Such remarkable cell penetrating ability may not only allow effective delivery of antimicrobials to the cells for intracellular pathogen clearance but also transport of other cargos, including proteins and nucleic acids.
  • the LPN can be a useful transport system for DNA/RNA to mammalian cells that are not easily transformable by the conventional liposome approach. Cytotoxicity
  • Example 7 Antibiotic-loaded PLGA/DOTAP particles inhibit bacteria in infected mammalian cells
  • the survival of intracellular S. aureus was determined after exposing the infected HaCaT cells to additional 250 pg/ml of free or LPN-encapsulated antimicrobials (i.e. Gentamicin, Vancomycin, Gentamicin+Vancomycin) for 20 h at 37 °C, 5% CO2.
  • the HaCaT cells were washed three times using PBS prior to cell lysis using 1 ml of PBS/triton-X (1 % v/v). The cell lysate was diluted 10x and plated on non-selective TSA agar. The plates were incubated at 37 °C for 24-48 h prior to enumeration. For comparison, a negative control was used, in which the infected HaCaT cells was not exposed to additional Gentamicin, Vancomycin or Gentamicin+Vancomycin (in either free-form or nanoparticle formulation).
  • the gentamicin protection 500 pg/mL was maintained throughout the experiment after the initial three-hour infection.
  • 250 pg/ml of antibiotics was the highest concentration used in the experiment to compare the treatment effectiveness between the free-from and the nanoparticle formulation. At this concentration (i.e., 250 pg/ml) or even as high as 500 pg/ml, the free-form antibiotics are not able to eliminate the intracellular bacteria.
  • T. pyriformis is a bacterial predator that is commonly used as an alternative to the macrophage model (i.e. a professional phagocyte for immune system).
  • the antibiotic used in this protocol was ampicillin, where the LPN was formed according to General Procedure 1 such that ampicillin is added at 10% w/w relative to the combined weight of the hydrophobic polymer and lipid.
  • T. pyriformis to high density of OG1 RF culture (i.e. 1x10 8 cells/ml) at a ratio of 1 : 10000 for a period of 8 h prior to the antibiotic treatment (i.e. free vs. LPN (PLGA/DOTAP) formulation).
  • the survival of T. pyriformis and OG1 RF were assessed at 20 h and 40 h post-antibiotic treatment ( Figure 1 1a).
  • Figure 1 1a For T. pyriformis enumeration, an aliquot of 100 pi well-mixed culture was sampled and examined under a light microscope. The sample was diluted sufficiently (1x-100x) to allow accurate counting of the surviving T. pyriformis.
  • T. pyriformis survival rate improved slightly to 15-25% at the highest antibiotic concentration examined (i.e. 250 pg/mL).
  • a similar survival rate of T. pyriformis could be readily achieved when as little as 50 pg/mL of ampicillin-encapsulated LPN was added.
  • LPN is an excellent drug carrier that is capable of penetrating animal cells for intracellular antimicrobial delivery and potentially, other cargo transport including nucleic acids, proteins etc.
  • LPNs were synthesized to co-encapsulate hydrophobic paclitaxel (PTX) and hydrophilic (-)- epigallocatechin gallate (EGCG), using General Procedure 1 , where 30 mg PLGA and a smaller quantity (1.0-3.3 mg) of DOTAP were dissolved in 1 mL acetone solvent. 3 mg PTX and 2 mg EGCG was then dissolved in the acetone solvent to provide a polymer solution.
  • PTX hydrophobic paclitaxel
  • EGCG hydrophilic epigallocatechin gallate
  • the PEG-lipid will form the outer layer (or form part of the outer layer) of the LPN and that this material enhances the colloidal stability of the LPN, while allowing the LPN to escape immunological recognition during systemic circulation;
  • a hydrophobic PLGA core was chosen to enable a high efficiency encapsulation of PTX and to delay its release relative to EGCG.
  • the drug-loaded LPNs were characterized for their physicochemical properties, as summarized in Table 6. FE-SEM micrographs found the synthesized LPNs to be spherical. Due to the DOTAP-lipid coating, LPNs were positively-charged with hydrodynamic sizes -115 nm.
  • LPNs were functionalized with hyaluronic acid (HA) and/or folic acid (FA), as targeting moieties to target CD44 and FA receptors, respectively.
  • Functionalized LPNs were prepared in the same way as the unfunctionalized LPNs made in this example except that a HA- or FA- conjugated DSPE-PEG-NH2were used in place of unmodified DSPE-PEG-NH2.
  • the carboxyl group of the HA or FA was conjugated with the DSPE-PEG2000-amine via EDC facilitated amide bond formation.
  • HA-conjugated DSPE-PEG (DSPE-PEG-NH-HA) was synthesized by coupling the DSPE- PEG-NH 2 with an activated HA [carboxyl group of HA (1 mmol) 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC; 1.2 mmol) in distilled water]. The reaction was performed at room temperature for 7 hours and then mixed with 50 mL of distilled water and centrifuged at 3000 rpm. After discarding the pellet, the supernatant was dialyzed and dried. The obtained polymer named DSPE-PEG-NH-HA was dried under vacuum overnight. A similar protocol was used to obtain FA-conjugated DSPE-PEG (DSPE-PEG-NH-FA) by analogy.
  • EDAC 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • EE Drug encapsulation efficiency
  • LPN an attractive vehicle for drugs of differing hydrophilicities, while maintaining a relatively high EE and avoiding drug- drug interactions.
  • surface modifying LPNs with FA- and/or HA-PEG-DSPE did not affect the EE of both drugs, confirming minimal drug loss through this facile surface modification process.
  • the cells were seeded into a 6-well plate at the density of 1 x 10 6 cells per well. After 24 h incubation, the cells were further incubated with different formulations at 5 nM of PTX concentrations at 37°C. After 4 h of incubation, the cell medium containing different formulations was discarded from the wells and the cells were washed with cold PBS. The cells were lysed for 10 min by addition of 1 % Triton X-100 (400 pL) per each well. After that, an aliquot of the cell lysate was used to measure the total cell protein amount by the BCA assay. The concentration of PTX in the cell lysate was measured by HPLC as described above.
  • the cells were incubated on glass base dish (Thermo scientific, USA) and were examined by confocal laser scanning microscopy (CLSM, Model: LSM5LIVE; Carl Zeiss, Wetzlar, Germany).
  • CLSM confocal laser scanning microscopy
  • MCF-7 cells were seeded at a density of 1 x 10 6 cells per well in 1 mL of growth medium and incubated for 24 h to allow them to attach. The cells were then treated with the different formulations in growth medium. After 4 h of incubation, the cells were washed with cold PBS. Then, cells were stained with 1 pg/mL DAPI in PBS for 3 min and washed with PBS. The cells were observed directly under the CLSM.
  • dual-targeted LPN was 1.6- and 1.9 folds higher than FA-targeted LPN and HA-targeted LPN, respectively.
  • the results strongly suggest the superior targeting ability of dual-targeted LPN as opposed to just single-targeted (HA or FA only) LPN.
  • 3D spheroid of MCF-7 cells were generated ( Figure 16), and treated with PTX/EGCG solution, FA-LPN, HA-LPN and HA-FA-LPN for 1 , 2 and 3 days.
  • MCF-7 cells were magnetically labeled using a previously established method (Ho VH, et al. Adv Healthc Mater. 2013;2: 1430-1434). MCF-7 cells were first incubated with 750 mM BiotinSE in PBS for 30 min. The biotinylated cells were mixed with 0.025 mg/mL streptavidin paramagnetic particles and vortexed for 15 s. Magnetically labeled cells at 1000 cell seeding density were dispensed into wells of 96-well round bottom low attachment plate (Corning Inc. 7007) in 100 pL medium per well.
  • the spheroids were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco 11965) supplemented with 10% fetal bovine serum, 100 pg.mL -1 penicillin, and 100 pg.mL -1 streptomycin. The spheroids were incubated in a 5% CO2 humidified atmosphere at 37°C.
  • DMEM Modified Eagle’s Medium
  • LPNs exerted a greater inhibitory effect on the spheroid growth than the free PTX/EGCG solution.
  • encapsulating EGCG and PTX in LPN enabled improved cytotoxicity against PTX
  • targeted LPNs exhibited significant higher intensity of red fluorescence over non- targeted LPN.
  • HA-FA-LPN was observed to elicit a continuous increased in the cell death over time, while single targeted LPN revealed no significant difference of fluorescence between day 2 and day 3.
  • HA-FA-LPN exhibited strong fluorescence all around spheroid while the other LPNs showed relative stronger intensity peaks on the outer rim of the spheroids than center region. This result indicated that the targeting effect of dual-targeted LPN gave rise to great cytotoxicity.
  • the initial cell death was not significant, dual-targeted LPN can also penetrate into the primed tumor, and cause substantial apoptosis.
  • the therapeutic efficiency of the different formulations was evaluated using MCF-7 human breast cancer-bearing mice.
  • the mice were treated with different formulations (5.0 mg PTX and 2.5 mg EGCG equiv./kg) when the tumor size was about 100 mm 3 in volume.
  • the treatment was given at day 0, 7 and 14.
  • the mice in the control group received PBS.
  • a solid tumor was established by subcutaneous injection of 0.1 ml_ MCF-7 cell suspension (1 c 10 6 cells per mouse) in the right flank of female nude mice.
  • Tumor growth was documented by measuring the length and width of the tumors using a Vernier Caliper twice weekly, and tumor volumes were calculated as length c width 2 /2. Intravenous injection was initiated when tumors reached a volume of ⁇ 100 mm 3 .
  • the rats were randomly divided into seven groups, and control, free drug control (free PTX, free PTX+EGCG), EGCG/PTX LPN, FA-EGCG/PTX LPN, HA- EGCG/PTX LPN, HA-FA-EGCG/PTX LPN (100 pL of 20 mg/kg PTX and 10 mg/kg EGCG) were intravenous injection 3 times (Day 0, 7, 14).

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Abstract

La présente invention concerne une nanoparticule de lipide-polymère destinée à être utilisée en administration de médicament. La nanoparticule contient une partie de cœur formée à partir d'un matériau de matrice polymère hydrophobe, entourée d'une partie enveloppe formée à partir de lipides et d'un tensioactif, les lipides contenant un lipide cationique et le rapport en poids:poids du lipide cationique au matériau de matrice polymère hydrophobe étant de 1:1 à 1:50. Dans un mode de réalisation préféré, le matériau de matrice polymère hydrophobe est du poly(acide glycolique-co-L,D-acide lactique) (PGLA) et le lipide cationique est le 1,2-dioléoyloxy-3-(triméthylammonium)propane (DOTAP). La présente invention concerne également des procédés de fabrication de la nanoparticule de lipide-polymère et l'utilisation de nanoparticules de lipide-polymère qui contiennent un principe pharmaceutique actif pour le traitement de maladies.
PCT/SG2019/050009 2018-01-05 2019-01-07 Nanoparticules hybrides de lipide-polymère WO2019135715A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020064844A1 (fr) * 2018-09-26 2020-04-02 Ascendis Pharma A/S Traitement d'infections
CN112168814A (zh) * 2020-10-28 2021-01-05 烟台大学 夫西地酸a环并氨基噻唑环衍生物的应用
CN112402394A (zh) * 2020-11-25 2021-02-26 天津医科大学第二医院 利用正负电荷特性吸附了纳米药物载体的卡介苗复合体及其制备方法
WO2021098621A1 (fr) * 2019-11-20 2021-05-27 深圳先进技术研究院 Nanoparticule avec immunoadjuvant encapsulé et son utilisation
CN113209044A (zh) * 2021-01-29 2021-08-06 广州医科大学 靶向递送HDACIs的仿生纳米颗粒及其制备方法和应用
CN114470226A (zh) * 2020-11-13 2022-05-13 中国科学技术大学 纳米颗粒包裹的抗生素及其制备方法和应用
CN114848594A (zh) * 2022-05-11 2022-08-05 南通大学 一种负载抗癌药物的脂质纳米载体及其制备方法与应用
WO2023240022A3 (fr) * 2022-06-06 2024-01-18 Suntec Medical, Inc. Conjugué pour cibler un système nerveux central

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016144766A1 (fr) * 2015-03-06 2016-09-15 The University Of North Carolina At Chapel Hill Metformine polymère et son utilisation comme agent thérapeutique et comme véhicule d'administration
CN107412191A (zh) * 2016-02-16 2017-12-01 烟台大学 一种长循环脂质-聚合物杂化纳米粒及其制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016144766A1 (fr) * 2015-03-06 2016-09-15 The University Of North Carolina At Chapel Hill Metformine polymère et son utilisation comme agent thérapeutique et comme véhicule d'administration
CN107412191A (zh) * 2016-02-16 2017-12-01 烟台大学 一种长循环脂质-聚合物杂化纳米粒及其制备方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BAEK, J.-S. ET AL.: "A programmable lipid-polymer hybrid nanoparticle system for localized, sustained antibiotic delivery to Gram-positive and Gram-negative bacterial biofilms", NANOSCALE HORIZONS, vol. 3, no. 3, 8 February 2018 (2018-02-08), pages 305 - 311, XP055619832 *
BOSE, R. J. C. ET AL.: "Lipid polymer hybrid nanospheres encapsulating antiproliferative agents for stent applications", JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY, vol. 36, 24 February 2016 (2016-02-24), pages 284 - 292, XP029521998, [retrieved on 20190102], doi:10.1016/j.jiec.2016.02.015 *
GAJRA, B. ET AL.: "Formulation and optimization of itraconazole polymeric lipid hybrid nanoparticles (Lipomer) using box behnken design", DARU JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 23, no. 1, 21 January 2015 (2015-01-21), XP021210575, [retrieved on 20190201], doi:10.1186/s40199-014-0087-0 *
GOSSMANN, R. ET AL.: "New Perspective in the Formulation and Characterization of Didodecyldimethylammonium Bromide (DMAB) Stabilized Poly(Lactic-co-Glycolic Acid) (PLGA) Nanoparticles", PLOS ONE, 6 July 2015 (2015-07-06), XP055619829 *
HU , Y. ET AL.: "Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability", ACTA BIOMATERIALIA, vol. 28, 30 September 2015 (2015-09-30), pages 149 - 159, XP055483700, [retrieved on 20190201], doi:10.1016/j.actbio.2015.09.032 *
LIU, L. ET AL.: "Hyaluronic Acid-Modified Cationic Lipid-PLGA Hybrid Nanoparticles as a Nanovaccine Induce Robust Humoral and Cellular Immune Responses", ACS APPL. MATER. INTERFACES, vol. 8, no. 19, 4 May 2016 (2016-05-04), pages 11969 - 11979, XP055619828 *
ZHANG, B. ET AL.: "Lung cancer gene therapy: Transferrin and hyaluronic acid dual ligand-decorated novel lipid carriers for targeted gene delivery", ONCOLOGY REPORTS, vol. 37, no. 2, 7 December 2016 (2016-12-07), pages 937 - 944, XP055619831 *
ZHAO, P. ET AL.: "Paclitaxel loaded folic acid targeted nanoparticles of mixed lipid-shell and polymer-core: In vitro and in vivo evaluation", EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, vol. 81, no. 2, 14 March 2012 (2012-03-14), pages 248 - 256, XP055451933, [retrieved on 20190201], doi:10.1016/j.ejpb.2012.03.004 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020064844A1 (fr) * 2018-09-26 2020-04-02 Ascendis Pharma A/S Traitement d'infections
WO2021098621A1 (fr) * 2019-11-20 2021-05-27 深圳先进技术研究院 Nanoparticule avec immunoadjuvant encapsulé et son utilisation
CN112168814A (zh) * 2020-10-28 2021-01-05 烟台大学 夫西地酸a环并氨基噻唑环衍生物的应用
CN114470226A (zh) * 2020-11-13 2022-05-13 中国科学技术大学 纳米颗粒包裹的抗生素及其制备方法和应用
CN114470226B (zh) * 2020-11-13 2024-02-23 中国科学技术大学 纳米颗粒包裹的抗生素及其制备方法和应用
CN112402394A (zh) * 2020-11-25 2021-02-26 天津医科大学第二医院 利用正负电荷特性吸附了纳米药物载体的卡介苗复合体及其制备方法
CN113209044A (zh) * 2021-01-29 2021-08-06 广州医科大学 靶向递送HDACIs的仿生纳米颗粒及其制备方法和应用
CN114848594A (zh) * 2022-05-11 2022-08-05 南通大学 一种负载抗癌药物的脂质纳米载体及其制备方法与应用
CN114848594B (zh) * 2022-05-11 2023-04-25 南通大学 一种负载抗癌药物的脂质纳米载体及其制备方法与应用
WO2023240022A3 (fr) * 2022-06-06 2024-01-18 Suntec Medical, Inc. Conjugué pour cibler un système nerveux central

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