US20110052633A1 - Multi-phase emulsions based on amphiphilic block copolymers - Google Patents

Multi-phase emulsions based on amphiphilic block copolymers Download PDF

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US20110052633A1
US20110052633A1 US12/553,086 US55308609A US2011052633A1 US 20110052633 A1 US20110052633 A1 US 20110052633A1 US 55308609 A US55308609 A US 55308609A US 2011052633 A1 US2011052633 A1 US 2011052633A1
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peg
poly
emulsion
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block
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Ming-hsi Huang
Pele Choi-Sing Chong
Chih-Hsiang Leng
Shih-Jen Liu
Hsin-Wei Chen
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National Health Research Institutes
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Assigned to NATIONAL HEALTH RESEARCH INSTITUTES reassignment NATIONAL HEALTH RESEARCH INSTITUTES ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHEN, HSIN-WEI, CHONG, PELE CHOI-SING, HUANG, MING-HSI, LENG, CHIH-HSIANG, LIU, SHIH-JEN
Priority to TW098130839A priority patent/TWI383806B/zh
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Priority to US13/604,610 priority patent/US8444993B2/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/145Orthomyxoviridae, e.g. influenza virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • A61P31/16Antivirals for RNA viruses for influenza or rhinoviruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/06Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/66Polyesters containing oxygen in the form of ether groups
    • C08G63/664Polyesters containing oxygen in the form of ether groups derived from hydroxy carboxylic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525Virus
    • A61K2039/5252Virus inactivated (killed)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55555Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55566Emulsions, e.g. Freund's adjuvant, MF59
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/16011Orthomyxoviridae
    • C12N2760/16111Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to emulsion formulations, and more specifically to a multi-phase emulsion formulations.
  • the emulsion-type adjuvants have the advantages of ease of manufacture and low costs.
  • Freund's adjuvants and Montanide® ISA 51 (ISA51), containing mineral oil and lipophilic emulsifier named mannide monooleate, are defined as water-in-oil (W/O) emulsions with dispersed antigenic media and continuous oily phases.
  • W/O water-in-oil
  • the W/O types of adjuvant products have been evaluated to improve the innocuity of the vaccine and to achieve long-term protective immune responses. It is difficult to give injections with syringe needle having a small diameter. There are also local reactions at the injection site, which limit their applications to humans.
  • Tween®80 polyoxyethylene sorbitan monooleate
  • LMWS low-molecular weight surfactants
  • TiterMax® in which a squalene-based water-in-oil (W/O) emulsion is stabilized by microparticulate silica and the non-ionic block copolymer polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE, known as Pluronic® or Poloxamer®).
  • W/O water-in-oil
  • POE-POP-POE polyoxyethylene-polyoxypropylene-polyoxyethylene
  • composition comprising:
  • the aforementioned composition is dispersed in a phosphate-buffered saline solution (PBS), wherein the composition is in the form of an oil-in-water (O/W) emulsion, and wherein the aqueous phase comprises PBS.
  • PBS phosphate-buffered saline solution
  • the oily phase may entrap or encapsulate an antigen and/or a bioactive agent.
  • the continuous aqueous phase may comprise an antigen and/or a bioactive substance.
  • the aforementioned composition is free of an organic solvent.
  • the oily phase further comprises an emulsion comprising:
  • the lipophilic emulsifying system comprises at least one physiologically acceptable emulsifier selected from the group consisting of mannide monooleat and sorbitan esters.
  • the W/O/W emulsion formulation may further comprise an antigen and/or a bioactive agent dissolved in the internal aqueous phase and/or in the continuous aqueous phase.
  • the antigen may be an inactivated virus, e.g., H5N1 virus, or a bacterium and/or an antigenic protein or an antigenic fusion protein.
  • the inner aqueous phase further comprises an antigen and/or a bioactive agent.
  • Another aspect of the invention relates to a block copolymer comprising the formula:
  • the hydrophilic block (B)q constitutes at least 50% by weight of the block copolymer.
  • p is 0 and A is methoxy.
  • the block copolymer is bioresorbable.
  • the hydrophilic block (B)q is a liner polymer.
  • the repeating unit B is selected from the group consisting of ethylene oxide, vinylpyroolidone, and acrylamide.
  • the hydrophobic blocks (A)p and (C)r are each polyester polymers.
  • hydrophobic blocks (A)p and (C)r are each aliphatic polyesters.
  • repeating units A and C are each selected from the group consisting of hydroxyacids, lactones, and combinations thereof.
  • the hydroxyacids may be selected from the group consisting of lactic acid, 6-hydroxycaproic acid, glycolic acid, malic acid monoesters, and combinations thereof.
  • the lactones may be selected from the group consisting of ⁇ -caprolactone, lactide, glycolide, para-dioxanones, and combinations thereof.
  • the aliphatic polyesters are polymers of dicarboxylic acids and a diols.
  • the hydrophobic blocks (A)p and (C)r are each comprises a polymer selected from the group consisting of the following:
  • the block copolymer is selected from the group consisting of poly(ethylene glycol)-block-poly(lactide-co-c-Caprolactone), poly(ethylene glycol)-block-polylactide, and poly(ethylene glycol)-block-poly( ⁇ -caprolactone).
  • FIG. 1 shows (a) the visual aspects of emulsions stored at 37° C. and (b) the cumulative release of OVA from the emulsion formulations based on bioresorbable polymers and the oily adjuvant ISA5 I (-x-) Non-formulation, (open circle) PEG-b-PLA/ISA51, (open square) PEG-b-PCUISA51, (open triangle) PEG-b-PLACL/ISA51, (filled circle) PBS/ISA51.
  • the OVA-containing formulations (3 mg per 0.3 mL) were placed in a dialysis chamber in a centrifuge tube containing 2 ml PBS and stood at 37° C.
  • the OVA release was regularly monitored by the BCA method (read by an UV-vis instrument at 562 nm, using calibration curves obtained from standard BSA solutions). The data are presented as the mean with standard deviation of three samples.
  • FIGS. 2( a )- 2 ( d ) are schematic presentations showing an ISA51-adjuvanted vaccine (a) before and (b) after stabilization with (c) a bioresorbable polymer.
  • the ISA51 contains lipophilic mannide monooleate, which renders a W/O emulsion because of its high affinity for the oily phase.
  • the water affinity of the oily ISA51-adjuvanted vaccine was greatly enhanced so that the antigen-encapsulated stock emulsion was able to be re-dispersed in the PBS and resulted in an emulsion with homogeneous fine particles having (d) a particle size distribution of ⁇ 1 ⁇ m.
  • FIG. 3 is a graph showing specific antibody responses in mice following immunization with OVA in different formulations.
  • BALB/c mice were subcutaneously vaccinated twice at weeks 0 and 2 with 0.5 ⁇ g of OVA.
  • Sera were collected from blood and the antibody titers were measured by ELISA. The data are presented as geometric mean titers with standard errors (5 mice per group). *P ⁇ 0.005: A comparison to the non-formulated OVA group was made at the same time point.
  • FIGS. 4( a )- 4 ( b ) show the schemes for synthesizing (a) the diblock copolymer PLA-PEG and (b) the triblock copolymer PLA-PEG-PLA.
  • FIGS. 5( a )- 5 ( b ) show the MALD1-TOF mass spectra of (a) MePEG 2000 and (b) PLA-PEG.
  • FIGS. 6( a )- 6 ( b ) show the MALDI-TOF mass spectra of (a) di0H-PEG 2000 and (b) PLA-PEG-PLA.
  • FIG. 7 is a graph showing the in vitro OVA release from the squalene emulsions based on the PLA-PEG and PLA-PEG-PLA.
  • the OVA-containing formulations (3 mg per 0.3 mL) were placed in a dialysis chamber in a centrifuge tube containing 2 ml of PBS and stood at 37° C. The release was regularly monitored by the RCA method and read by an UV-vis instrument at 562 nm using calibration curves obtained from standard BSA solutions. Data are presented as the mean with standard errors of three samples.
  • (open triangle) PLA-PEG-PLA/squalene (open triangle) PLA-PEG-PLA/squalene.
  • FIGS. 8( a )- 8 ( c ) are photographs showing polymer/oil emulsions (a) before and (b) after homogenization at 6000 rpm for 5 min, and (c) the visual aspects of the emulsions stored at 4° C. for two months.
  • the polymer concentration in the antigen medium aqueous solution was 13 wt % and the aqueous/oily solution was 5/5 w/w.
  • PEG-b-PLACL/squalene was 13 wt % and the aqueous/oily solution was 5/5 w/w.
  • PEG-b-PLACL/squalene PEG-b-PLACL/squalene/Span®85
  • FIGS. 9( a )- 9 ( b ) are photographs showing the results of droplet tests of the emulsion formulations (a) PBS/squalene/Span®85 and (b) PEG-b-PLACL/squalene.
  • FIGS. 10( a )- 10 ( b ) show microscopic aspects and laser light scattering analysis of polymer-emulsified formulations (a) PEG-b-PLACL/squalene and (b) of PEG-b-PLACL/squalene/Span®85.
  • FIG. 11( a ) is a graph showing the cumulative release of OVA from various formulations.
  • (-x-) No adjuvant, (open circle) the aqueous solution PEG-b-PLACL, (open square) the O/W emulsion PEG-b-PLACL/Squalene, (open triangle) the W/O/W emulsion PEG-b-PLACL/squalene/Span®85, (filled circle) the W/O emulsion PBS/squalene/Span®85.
  • the OVA-containing formulations (3 mg per 0.3 mL) were placed in a dialysis chamber in a centrifuge tube containing 2 mL of PBS and stood at 37° C.
  • the OVA release was monitored by the BCA method and read by an UV—vis instrument at 562 nm using calibration curves obtained from the standard BSA solutions (2, 1, 0.5, 0.25, 0.125 mg/mL). The data are presented as the mean with standard errors of three samples.
  • FIG. 11( b ) is photograph showing the recovered formulations after the experiments in FIG. 11( a ).
  • FIGS. 12( a )- 12 ( b ) show (a) T-cell proliferation and (b) cytokine release responses in spleen cells from mice immunized with OVA in different formulations with or without adjuvants.
  • control (i) no adjuvant;
  • PEG-b-PLACL/squalene (iv) PEG-b-PLACL/squalene/Span®85;
  • aluminum phosphate aluminum phosphate.
  • the BALB/c mice were vaccinated subcutaneously at week 0 with 0.5 ⁇ g of OVA and boosted at weeks 2 and 4.
  • splenocyte suspensions from a pool of two mice per group were prepared for cytokines assay and incubated over five days with or without 10 ⁇ g/mL of antigen OVA.
  • FIG. 13 is a schematic representation of a W/O/W emulsion.
  • the emulsion PEG-b-PLACL/squalene/Span®85 comprises two surfactants, PEG-b-PLACL and Span®85, rendering a W/O/W multi-phase emulsion, in which the oil droplets were dispersed in the continuous water, but the core oil also traps an internal aqueous phase.
  • FIG. 14 shows a PELC-formulated influenza vaccine having homogeneous fine particles with diameters ranging from 200 to 400 nm.
  • the inset is a histogram showing the particles' size distribution.
  • FIG. 15A is a graph showing T-cell proliferation in the splenocytes of mice immunized with inactivated H5N1 virus alone or with PELC.
  • the BALB/c mice were vaccinated i.m. with a single-dose of 0.5 ⁇ g of viral HA. Twelve days after immunization, splenocyte suspensions were pooled from three mice per group and incubated for four clays with or without 0.25 ⁇ g HA/mL of antigen.
  • FIG. 15B is a graph showing cytokines release from the splenocytes.
  • “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
  • immuno adjuvants shall generally mean products which increase the reactions of the immunity system when they are administered in the presence of antigen of virus, bacterial or synthetic origin.
  • biodegradable shall generally mean solid polymeric materials which break down due to macromolecular degradation with dispersion in vivo but no proof for the elimination from the body (this definition excludes environmental, fungi or bacterial degradation).
  • Biodegradable polymeric systems can be attacked by biological elements so that the integrity of the system, and in some cases but not necessarily, of the macromolecules themselves is affected and gives fragments or other degradation by-products. Such fragments can move away from their site of action but not necessarily from the body.
  • bioresorbable shall generally means solid polymeric materials which show bulk degradation and further resorb in vivo; i.e. polymers which are eliminated through natural pathways either because of simple filtration of degradation by-products or after their metabolization. Bioresorption is thus a concept which reflects a total elimination of the initial foreign material and of bulk degradation by-products (low molecular weight compounds) with no residual side effects.
  • bioresorbable assumes that the elimination is shown conclusively (Dietmar W. Hutraum (2000) “Scaffolds in tissue engineering bone and cartilage” Biomaterials 21:2529-2543, which is herein incorporated by reference in its entirety).
  • a “lactide” is a cyclic diester of lactic acid, i.e., a di-lactone.
  • a “glycolide” is a cyclic diester of glycolic acid, which is also a di-lactone.
  • dicarboxylic acids are organic compounds that are substituted with two carboxylic acid functional groups, such as succinic acid.
  • a “diol” or “glycol” is a chemical compound containing two hydroxyl groups (—OH groups), such as ethylene glycol.
  • a “hydroxy acid” is an organic compound which contains a carboxylic acid functional group and hydroxy functional group.
  • amphiphile means any organic compounds composed of hydrophilic and hydrophobic portions.
  • the term “the ratio q/(p+r) is sufficient high” means that the block copolymer of the formula of (A)p-(13)q-(C)r has a hydrophilic-lipophilic balance (HLB) greater than 10 so that it promotes an oily phase to disperse in an aqueous phase and result in an oil-in-water (O/W) emulsion.
  • HLB hydrophilic-lipophilic balance
  • the HLB of the amphiphilc block copolymer may be greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, or near 20.
  • the term “A is other than a reactive functional group or a reactive group” refers to any group that does not react under conditions where the non-protected group reacts.
  • the group “A” protects reactive functional groups, such as hydroxyl or amino groups, from their reaction with growing species in polymerization.
  • hydroxy-protecting group refers to any group commonly used for the protection of hydroxy functions during subsequent reactions, including, for example, alkoxyl, acyl or alkylsilyl groups such as trintethylsilyl, triethylsilyl, t-butyldimethylsilyl and analogous alkyl or arylsilyl radicals, or alkoxyalkyl groups such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, tetrahydrofuranyl or tetrahydropyranyl.
  • a “protected-hydroxy” is a hydroxy function derivatized by one of the above hydroxy-protecting groupings.
  • Alkyl represents a straight-chain or branched hydrocarbon radical or 1 to 10 carbons in all its isomeric forms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, etc., and the terms “hydroxyalkyl,” “fluoroalkyl” and “deuteroalkyl” refer to such an alkyl radical substituted by one or more hydroxy, fluoro or deuterium groups, respectively.
  • acyl group is an alkanoyl group of 1 to 6 carbons in all its'isomeric forms, or an aroyl group, such as benzoyl, or halo-, nitro-, or alkyl-substituted benzoyl groups, or an alkoxycarbonyl group of the type Alkyl--O—CO—, such as methoxycarbonyl, ethoxycarbonyl, propyloxycarbonyl, etc., or a dicarboxylic acyl group such as oxalyl, malonyl, succinoyl, glutaroyl, or adiopoyl.
  • aryl signifies a phenyl-, or an -alkyl-, nitro-, or halo-substituted phenyl group.
  • alkoxy signifies the group, alkyl-O—.
  • Emulsifiers may be defined by their hydrophilic-lipophilic balance (HLB) values, which give information on their relative affinity for aqueous and oily phases.
  • HLB hydrophilic-lipophilic balance
  • An emulsifying system which contains an emulsifier of low HLB value renders a W/O emulsion with a high affinity for an oily phase.
  • an emulsifying system which contains a high HLB value affords an O/W emulsion with a high affinity for an aqueous phase.
  • a W/O/W multi-phase emulsion may be achieved when an emulsifying system contains an intermediate HLB value.
  • the HLB is calculated based on the following equation:
  • HLB mix ⁇ X i ⁇ HLB i
  • HLB For non-ionic surfactants, HLB may be calculated with the Griffin's method:
  • M h is the molecular mass of the hydrophilic portion of the molecule, and M is the molecular mass of the whole molecule.
  • the HLB of the most lipophilic molecule is close to 0, while the HLB of the most hydrophilic molecule is about 20.
  • HLB copolymer 20 ⁇ W h /W copolymer
  • W h /W copolymer is the weight ratio of the hydrophilic portion of the main chain polymer and is obtained from their number average molecular weight ratio Mn h / Mn copolymer .
  • a W/O emulsion based on lipophilic mannide monooleate and water-immiscible oil has been available.
  • the oil used is the mineral oil Markol (Freund's adjuvants), metabolizable mineral oil Drakeol (Montanide® ISA 51) or metabolizable nonmineral squalene (Montanide® ISA 720).
  • TiterMax® is a squalene-based W/O emulsion stabilized by microparticulate silica and the nonionic block copolymer polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE, known as Pluronic® or Poloxamer®).
  • the invention relates to formulations for enhancing the water affinity oil-in-water emulsions, such as oily ajuvanted vaccines.
  • the HLB is sufficient high so that the block copolymer is capable of stabilizing the interface between an oil phase and an aqueous phase and promotes the dispersion of the oil phase into the aqueous phase and forms a stable O/W emulsion. This is particular useful in redispersing an ISA51-adjuvanted oily vaccine, a W/O emulsion, into a PBS to form a W/O/W multiphase emulsion.
  • q is an integer and the hydrophilic portion (B)q has a molecular weight of from about 550 to about 10,000 daltons, preferably from about 2,000 to about 8,000 daltons, and the ratio q/(p+r) is sufficient high so that the hydrophilic portion (B)q constitutes from about 50 to about 95% by weight of the main chain polymer (A)p-(B)q-(C)r, which renders the hydrophilic-lipophilic balance (HLB) of the block copolymer ranging from about 10 to about 19.
  • HLB hydrophilic-lipophilic balance
  • the hydrophilic portion (B)q constitutes from about 70 to about 95% by weight of the main chain polymer so that the HLB value ranges from about 14 to about 19.
  • the ratio of q versus p+r is important for the HLB of amphiphilic macromolecules. If q/(p+r) is not high enough, then the macromolecules do not dissolve in water.
  • Tables A and B list the block copolymers' molar ratios, q/(p+r), and HLB values (See Huang et al. (2009) “Development of Multi-Phase Emulsions Based on Bioresorbable Polymers and Oily Adjuvant” Pharmaceutical Research 26(8): 1856-1862; Huang et al., (2004) Degradation and cell culture studies on block copolymers prepared by ring opening polymerization of ⁇ -caprolactone in the presence of poly(ethylene glycol). J.
  • Oil must be non-toxic, metabolizable, physiologically acceptable, and form fluid emulsions when stored at 4° C.
  • Oils are selected from mineral oils, vegetable or animal oils known for low toxicity.
  • the selected mineral oils may be straight chain mineral oils, e.g., Markol (Freund's adjuvants) or Drakeol (Montanide® ISA 51).
  • Synthetic hydrocarbons include polyisobutene and polyisoprene.
  • Suitable vegetable oils include oleic type unsaturated oils that are biodegradable and known for immunogenic power, such as ground-nut oil, olive oil, sesame oil, soya bean oil, corn oil, and jojoba oil, etc.
  • Suitable animal oils require the same criteria of tolerance and immunological efficiency, such as squalane and squalene (MF59®, AS03, Montanide® ISA 720, TiterMax).
  • the polymer blocks (A)p and (C)r are hydrophobic linear polyester blocks, e.g., aliphatic polyesters.
  • Aliphatic polyesters may be obtained as follows: a) either by self-polycondensation of a hydroxyacid (i.e., homopolymers), or by polycondensation of different hydroxyacids (i.e., heteropolymers), b) by the polymerization via ring-opening of lactones; and c) by polycondensation of diacids and diols.
  • Hydroxyacid monomers may be chosen from lactic acid, glycolic acid, 6-hydroxycaproic acid, malic acid monoesters, e.g., alkyl or aralkyl monoesters, or monoesters resulting from the monoesterification of mane acid with a hydroxylated active compound, in particular a hydrophobic active compound; lattides (D-lactide, L-lactide, DL-lactide, and meso-lactide), glycolide, ⁇ -caprolactone, para-dioxanone, and the like.
  • the hydrophobic polymer blocks (A)p and (C)r may be copolymers formed by the polymerization of different monomers.
  • the optimal length of (A)p and (C)r chains may be determined.
  • the polymer was added to PBS in the presence of the water-insoluble dye diphenylhexatriene, which would dissolve in the hydrophobic core of polymeric micelles or aggregates. After sonication and centrifugation, we observed an abrupt enhancement in the ultraviolet (356 nm) absorption of the dye, which indicated micelle formation.
  • the polymeric emulsifiers of the invention exhibit distinguishing features. They are biodegradable and bioresorbable. Degradation studies on these copolymers have shown that the polyester chains (A)p and (C)r are gradually degraded by hydrolysis. The final products are corresponding hydroxyacids (or diacids and diols), which are bioresorbable. Continuing hydrolysis will eventually release the hydrophilic polyester chain (B)q, the central block of the copolymer.
  • Such polymers of relatively low molecular mass (less than 10,000) are bioresorbable and may be excreted from the kidney.
  • the GSK ASO3 adjuvant which contains is prepared via a two-step manufacture process Tween®80, and alpha-tocopherol in a fluid O/W emulsion. It however lacks an HLB value.
  • the invention in another aspect, relates to a process for making a multi-phase W/O/W emulsion, which may trap and/or encapsulate antigens and/or bioactive substances in the multi-phase emulsion.
  • the process comprises a homogenizing (or called emulsifying) step and a diluting (or called dispersing) step.
  • a pre-emulsified stock comprising an oil and emulsifiers is obtained, in which a designed block copolymer serves as a hydrophilic emulsifier to stabilize an oil-water interface, and a lipophilic emulsifier serves to stabilize a water-oil interface and result in a stable and isotropic W/O/W emulsion.
  • the W/O/W multi-phase emulsion is achieved with an emulsifying system containing the designed block copolymer with an intermediate HLB value.
  • the oil droplets are dispersed in a continuous aqueous phase, in which the oil-water interface is stabilized by the block copolymer (HLB>>10).
  • the core oil entraps an aqueous phase, in which the entrapped water-oil interface is stabilized by a lipophilic emulsifier (HLB ⁇ 10).
  • the above pre-emulsified stock was diluted by redispersing it into an aqueous solution.
  • the aqueous solution may be an aqueous medium alone, such as PBS, or an aqueous medium containing an antigen or a bioactive substance such as peptides, anticancer agents, hormones, or other active agents such as antibiotics or antiparasitics.
  • the antigen or the bioactive substance may help disolving in either the oily or the aqueous phase.
  • a either in the internal and/or external aqueous phase of the release formulation, dissolving and encapsulating an has the effect of protecting the antigen. Conversely has the effect of facilitating the
  • hydrophilic polymeric emulsifiers namely, poly(ethylene glycol)-block-polylactide (PEG-b-PLA), poly(ethylene glycol)-block-poly( ⁇ -caprolactone) (PEG-b-PCL), and poly(ethylene glycol)-block-poly(lactide-co- ⁇ -caprolactone) (PEG-b-PLACL) in the antigen medium to alter the water affinity of oily ISA51-adjuvanted vaccines.
  • PEG-b-PLA poly(ethylene glycol)-block-polylactide
  • PEG-b-PLACL poly(ethylene glycol)-block-poly(lactide-co- ⁇ -caprolactone)
  • Tin(II) 2-ethylhexanoate (stannous octoate, SnOct 2 ) was purchased from Sigma (St. Louis, Mo., USA).
  • DL-lactide (a cyclic di-ester of lactic acid) was purchased from Aldrich (Seelze, Germany) and recrystallized from ethyl acetate.
  • ⁇ -Caprolactone was purchased from Aldrich.
  • Poly(ethylene glycol) 5,000 monomethyl ether (MePEG 5000 ) was purchased from Fluka (Buchs, Switzerland). All solvents were of analytical grade.
  • PEG-b-PLACL was synthesized by ring-opening polymerization of lactide and ⁇ -caprolactone, using SnOct 2 as a catalyst and MePEG 5000 as an initiator. Briefly, a predetermined amount of MePEG 5000 (2.1 g), lactide (0.58 g), and ⁇ -caprolactone (0.47 g) were placed in a dried round-bottomed bottle, and an appropriate amount of SnOct 2 (30 mg) was added as a solution in dried toluene (10 mL). Polymerization was performed at 140° C. under reflux for 24 hr. The product was recovered by precipitation in an excessive amount of ethanol.
  • PEG-b-PLA or PEG-b-PCL was synthesized in the same manner with MePEG/lactide or MePEG/ ⁇ -caprolactone at a weight ratio of 2/1.
  • the resulting polymers were characterized by 1 H nuclear magnetic resonance ( 1 H NMR) and gel permeation chromatography (GPC). 1 H NMR spectra were recorded at room temperature with a Varian VXR 300 MHz spectrometer (Varian, Palo Alto, Calif., USA) using deuterated chloroform as the solvent.
  • GPC was performed by using a setting composed of a Waters 510 HPLC pump, a Waters 410 differential refractometer, one PLgel mixed-C 5 ⁇ m 100 ⁇ column (7.5 ⁇ 300 mm), and one PLgel 3 ⁇ m 100 ⁇ column (7.5 ⁇ 300 mm), and the mobile phase being tetrahydrofuran (THF) and the flow rate 0.8 mL/min.
  • THF tetrahydrofuran
  • the antigen medium was prepared with a particular concentration of ovalbumin (OVA, Grade V, Sigma, St. Louis, Mo., USA) diluted in a phosphate-buffered saline (PBS).
  • An aqueous solution containing 120 mg of polymer and 0.8 mL of antigen medium and an oil solution containing 1.1 mL of ISA51 (Montanide® ISA 51 F VG, SEPPIC, Paris, France) were emulsified using a Polytron® PT 3100 homogeniser (Kinematica AG, Swiss) under 6,000 rpm for 5 min.
  • the stability test was performed by placing each sample at 4° C. and 37° C., and then noted the visual aspect at a predetermined time.
  • the droplet test was assessed by placing a droplet (20 ⁇ L) of an emulsion into the water in a beaker (200 mL).
  • the particle size distribution was determined by using the laser light scattering technique with a Brookhaven 90 plus particle sizer (Brookhaven Instruments Limited, New York, USA).
  • In vitro release experiments were performed by using the inverted dialysis tube method.
  • OVA-containing formulations (3 mg per 0.3 mL) were first placed in a dialysis chamber (cutoff 0.2 ⁇ m) and then the device was immersed in a 50 mL centrifuge tube containing 2 mL of PBS at 37° C. At different time intervals, 100 ⁇ L of sample were aspirated from the medium outside of the chamber and replaced with 100 ⁇ L of PBS buffer. The OVA release was regularly determined by the bicinchinonic acid method (BCATM protein assay kit, Pierce, Rockford, Ill., USA).
  • mice Five-week old female BA Laic. mice were obtained from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) and acclimatized for at least one week at the animal facility of National Health Research Institutes (NHRI, Miaoli, Taiwan) prior to use. All animal studies were approved by the Animal Committee of NHRI. Mice were primed subcutaneously (s.c.; 100 ⁇ L) with a syringe needle of 27G ⁇ 1 ⁇ 2′′ and 0.5 ⁇ g of OVA in PBS or formulated with PEG-b-PLACL/ISA51 or PBS/ISA51, and boosted with the same formulation at week 2.
  • s.c. 100 ⁇ L
  • PEG-b-PLACL/ISA51 was investigated by re-dispersing 100 ⁇ L of stock emulsion (See “MATERIALS AND METHODS” SECTION: Polymer-stabilized emulsions) into 900 ⁇ L of PBS before injection, resulting in a PEG-b-PLACL/ISA51/PBS emulsion of only 5% oil solution.
  • mice were bled at the lateral tail vein and the collected sera were stored at ⁇ 30° C.
  • the presence of OVA-specific antibodies in the sera was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 100 ⁇ L of diluted OVA (10 ⁇ g/mL) were coated onto 96-well microtiter plates with 0.05 M carbonate buffer (pH 9.6). After the overnight incubation at 4° C., coated plates were washed twice with PBS containing 0.05% Tween® 20 (Sigma, St. Louis, USA) and then blocked with 5% non-fat milk in PBS at room temperature for 2 hr.
  • ELISA enzyme-linked immunosorbent assay
  • Diluted sera (starting dilution 1:50, serial three-fold serum dilutions) from immunized animals were applied to wells at room temperature for 2 hr.
  • HRP-conjugated goat anti-mouse IgG ICN Cappel, Aurora, Ohio, USA
  • the assay was developed with a substrate solution containing tetramethylbenzidine (TMB, Sure BlueTM, KPL, MD, USA), and the reaction was stopped in 2 N H 2 SO 4 . Plates were read at 450 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, Calif., USA).
  • the titers were determined based on the reciprocal of the final dilution that gave 2-fold greater absorbance than the pre-immune sera. For isotype determination, 100 ⁇ L of an appropriate dilution (1:2,000) of HRP-rabbit anti-mouse IgG1 (Zymed®, Calif., USA) or HRP-rabbit anti-mouse IgG2a (Zyme®, Calif., USA) was added. Statistical significance (p ⁇ 0.005) was determined by performing two-tailed Student's t-test on log-transformed values.
  • AB-type diblock copolymers consisting of a polyether block (PEG) and a polyester block (PLA, PCL or PLACL) were synthesized by ring-opening polymerization of lactide and/or ⁇ -caprolactone in the presence of MePEG, using SnOct 2 as a catalyst.
  • the molecular characteristics of the three copolymers are summarized in Table 1.
  • MePEG with a molecular weight of 5,000 and an initial hydrophilic/lipophilic ratio of 2/1 were selected as a compromise between the high hydrophilicity of polymers and the bioresorbability of PEG-rich degradation products.
  • PEG is a water-soluble polymer, particularly, low molecular weight PEG ( ⁇ 10,000 Daltons) can be excreted through kidney filtration.
  • the lipophilic block was derived from the U.S. Food and Drug Administration (FDA)-approved aliphatic polyesters, PLA and PCL. They show bulk degradation and further resorb in vivo. PLA with a variable chain stereoregularity provides a worthwhile means to adjust the rate of degradation.
  • the degradation products of PCL had a relatively higher pKa than those of poly(lactide-co-glycolide) (4.8 for ⁇ -hydroxycaproic acid, and 3.8 for lactic acid and glycolic acid at 25° C.), and they may provide more conservation of protein molecular integrity when being used for a long-term controlled delivery of proteins.
  • PLACL was chosen as the lipophilic block for its fast degradation characteristics.
  • its amorphous nature provides a good affinity between the polymer matrix and oil solutions.
  • SnOct 2 has been approved by the U.S. FDA for biomedical and therapeutic applications.
  • the molar ratio of lactyl units to caproyl units to oxyethylene or [LA]:[CL]:[OE] was determined from the integrations of the proton resonances due to PLA blocks at 5.2 ppm, to PCL blocks at 4.1 ppm, and to PEG blocks at 3.6 ppm on the 1 H NMR spectra.
  • the number average molecular weight ( Mn ) were calculated according to the following equation:
  • HLB hydrophilic-lipophilic balance
  • HLB copolymer 20 ⁇ ( W PEG W copolymer )
  • W PEG /W copolymer is the weight ratio of the hydrophilic portion of the main chain polymer and is obtained from Mn PEG / Mn copolymer .
  • the most lipophilic polymer has an HLB number approaching 0, and the most hydrophilic polymer has a HLB of about 20.
  • HLB PEG-b-PLA of 14.4, HLB PEG-b-PCL of 14.0, and HLB PEG-b-PLACL of 15.0
  • hydrophilic PEG-b-PLA, PEG-b-PCL, and PEG-b-PLACL hydrophilic PEG-b-PLA, PEG-b-PCL, and PEG-b-PLACL as emulsifiers to stabilize the interface between the ISA51 oily adjuvant and the antigen media.
  • An aqueous phase of polymer dissolved in antigen media and an oily phase of ISA51 were emulsified using a homogenizer.
  • the emulsifying formulation was perfectly white and isotropic from the top to the bottom.
  • the stability test was investigated at 4° C. and at 37° C. During the storage at 4° C., all emulsions were stable for a few weeks without phase separation. In the case of PEG-b-PLA/ISA5 I and PEG-b-PCL/ISA5I, 10% of water disassociated after two weeks, but beyond this, no further water disassociation from the emulsion occurred. An isotropic emulsion could be re-formed by vortex mixing. On the other hand, approximately 10% of free oil at the surface layer disassociated from the PBS/ISA51 emulsion after one month under the same storage conditions.
  • the PEG-b-PLACL/ISA51 emulsion was stable for at least six months without phase separation. During 60 days' monitoring at 37° C., the PEG-b-PCL/ISA5I and PEG-b-PLACL/ISA51 emulsions were stable without phase separation. On the other hand, approximately 10% of free oil at the surface layer disassociated from the PBS/ISA51 emulsion after 3 days ( FIG. 1 a ). After one week, 30% of free oil at the surface and clear layers of water (30%) at the bottom disassociated from the emulsion. Phase separation happened at day 60, indicating the emulsion breaks. In the case of PEG-b-PLA/ISA51, the change in the visual aspect over time was similar to the PBS/ISA51.
  • the water affinity of the emulsions was investigated by the droplet test and laser light scattering.
  • an emulsified PBS/ISA51 droplet (arrow in FIG. 2 a inset) kept on floating on the water surface after 24 hr.
  • the particle size was not detected by using light scattering technology.
  • homogeneous particles with the size distribution of 1 ⁇ m were observed by optical microscope when re-dispersing the emulsion in the ISA51 oil solution (data not shown).
  • each polymer-stabilized ISA51 droplet (arrow in FIG. 2 b inset) could stand only for seconds in the aqueous phase and then diffuses in the water, which indicated its high affinity for water ( FIG. 2 b ).
  • the dynamic light scattering pattern showed that PEG-b-PLA, PEG-b-PCL or PEG-b-PLACL was a suitable emulsifier for the ISA51/water interface, and yielded narrowly distributed nanoparticles ( FIG. 2 d and Table 1). Typically, a bimodal distribution with two different sizes was observed, the relatively large particles of 500 nm and smaller ones of 100 nm ( FIG. 2 d ). This dimension is appropriate for uptake by antigen-presenting cells (APCs) to facilitate the induction of potent immune responses due to the pseudo-natural targeting of antigens. Homogenization using MePEG failed to improve the water affinity of ISA51. emulsion. The droplet floated on the surface instead o f diffusion in the water, which indicated that only PEG bearing short lipophilic units in the main chain polymer exhibited emulsifier property ( FIG. 2 c ).
  • APCs antigen-presenting cells
  • PEG-b-PLA-, PEG-b-PCL-, or PEG-b-PLACL-stabilized ISA51 emulsion also provided different controlled-release profiles to hydrophilic OVA protein with respect to free OVA or PBS/ISA51 -formulated OVA, as shown in FIG. 1 b .
  • a fast release was observed in the case of OVA without formulation from which more than 50% of loaded OVA was released into the outside PBS medium within the first 30 hr.
  • the PEG-b-PLA-modified ISA51 emulsion has similar release profiles to free OVA, in which less than 30% of OVA were released during the same period of time.
  • the protein release increased continuously until it reached the equilibrium concentration in the inside and outside of the dialysis device.
  • the oily PBS/ISA51 emulsion presented well depot effect to OVA so that hydrophilic OVA was slowly released over 500 hr.
  • the hydrophilic bioactive agents (or antigens) trapped within the PEG-b-PCL- or PEG-b-PLACL-stabilized emulsion will be released mostly by diffusion from the core oil to the surface, but also to a lesser extent by degradation mechanisms and emulsion breaks.
  • an ISA51 oily adjuvant contains only emulsifier of low HLB value (2.6 to mannide monooleate 202 , in which the hydrophobic end 204 facing toward the oil phase, and the hydrophilic end 206 facing toward the aqueous phase).
  • the water affinity test and in vitro release showed that the resulting PBS/ISA51 emulsion has a continuous phase of oil and the dispersed phase being water ( FIG. 2 a ).
  • a polymer-stabilized ISA51 system composes of two surfactants, hydrophilic polymer 208 and mannide monooleate 202 , rendered a water-in-oil-in-water (W/O/W) multi-phase emulsion ( FIG. 2 b ).
  • oil droplets dispersed into the continuous water stabilized by the polymeric emulsifier 208
  • the core oil also entrapped an aqueous phase (stabilized by mannide monooleate 202 ; FIG. 2 b ).
  • the polymer-emulsified particles could serve as either carriers or vehicles to deliver antigens to APCs in a targeted and prolonged manner.
  • the vaccine emulsions met the requirements for in vitro storage and the post-injection depot. It is generally recognized that oil droplets with small particle size and homogeneous distribution are more stable. These parameters are however strongly influenced by the optimization of the emulsification process and the surfactant system. To this end, addition of excipients like glycine or glycylglycine in Montanide® ISA 720, an oily adjuvant containing squalene and mannide monoolete, provided a potential way of stabilizing the emulsions during storage and post-injection.
  • excipients like glycine or glycylglycine in Montanide® ISA 720, an oily adjuvant containing squalene and mannide monoolete
  • MF59® developed from Novartis
  • AS03 developed from GlaxoSmithKline
  • MF59® is accomplished by using a combination of a hydrophilic Tween®80 emulsifier and a lipophilic Span®85 (sorbitan trioleate) while AS03 is stabilized by Tween®80 and alpha-tocopherol.
  • PEG-b-PLA-stabilized ISA51 emulsion remained the same or reduced the stability intrinsic to ISA51 oily adjuvant.
  • homogenization of PEG-b-PCL- or PEG-b-PLACL- containing aqueous solution and the mannide monooleate-contained oily phase provides a potential way of stabilizing the emulsified particles both at storage and at post-injection stage conditions.
  • the antibody assays were performed by subcutaneous vaccination in BALB/c mice with OVA, alone or formulated with PBS/ISA5I or PEG-b-PLACL/ISA51/PBS.
  • the latter contained only 5% of ISA51 oily adjuvant in the formulation (See “MATERIALS AND METHODS” section: Immunization and ELISA immunoassay).
  • the serum antibody IgG, IgG1 and IgG2a titers were significantly enhanced for the groups of PBS/ISA51 - and PEG-b-PLACL/ISA51/PBS- formulated OVA in comparison with the group of OVA alone (p ⁇ 0.005).
  • PEG-b-PLACIASA51/PBS-formulated OVA induced the same level of antibody titers as those induced by PBS/ISA51-formulated OVA.
  • the PEG-b-PLACL/ISA51/PBS reserved the adjuvant effects of PBS/ISA51.
  • vaccine antigens may be directly taken up by APCs, bind to the surface antibody on B cells, or undergo degradation. Only those antigens that are taken by APCs can integrate into the immune responses. The pathway is largely dependent on the characteristics of the antigen, but may also be influenced by the presence of adjuvants. Although emulsion-type adjuvants have been widely used for several decades, their immunogenicity-enhancing effects are still controversial due to the lack of understanding about the complexity of colloidal dispersions, the emulsion stability of post-injection and the mechanism of the immune response.
  • the adjuvant effect of the W/O emulsion could be explained by the depot of emulsion that is capable of slowly releasing antigen over a long period of time.
  • the W/O/W emulsion PEG-b-PLACL/ISA51/PBS emulsion as an example
  • W/O/W not only reserved the depot effects intrinsic to ISA5 I oil, but also combined the antigen presentation effects.
  • the ameliorated W/O/W emulsion increases injectability and conceptually diminishes local reactions with respect to the W/O type vaccines produced from the same oil.
  • Amphiphilic copolymers consisting of 70 wt-% hydrophilic PEG block and 30 wt-% lipophilic PLA, PCL or PLACL block were synthesized by the ring-opening polymerization of lactide and/or ⁇ -caprolactone on monomethoxy PEG 5000 .
  • the resulting polymers can serve as a hydrophilic emulsifier to alter the water affinity of oily ISA51-adjuvanted vaccines so that the stock antigen-encapsulated emulsion could be re-dispersed into PBS before injection and thus resulting in stable and injectable W/O/W emulsion nanoparticles.
  • the example here illustrates the synthesis and characterization of PEG-bearing PLA di- and tri-block copolymers, PLA-PEG and PLA-PEG-PLA, prepared by the direct polycondensation of an aqueous lactic acid solution on monomethoxy or dihydroxyl PEG.
  • PLA-PEG and PLA-PEG-PLA prepared by the direct polycondensation of an aqueous lactic acid solution on monomethoxy or dihydroxyl PEG.
  • block copolymers reported in the literature, which were prepared in the presence of cytotoxic catalyst-containing heavy metals, in this study, no catalyst was added during polymerization, which increased the confident biocompatibility in the final material.
  • the resulting copolymers were characterized by MALDI-TOF MS, GPC, and 1 H-NMR.
  • squalene was selected as the core oil because it has a low toxicity and is used in the clinical trial (Siao et al., (2009) “Characterization and Emulsifying Properties of Block Copolymers Prepared from Lactic Acid and Poly(ethylene glycol)” Journal of Applied Polymer Science 114: 509-516, which is incorporated by reference in its entirety).
  • Lactic acid was purchased as a 85-90% aqueous solution from TEDIA (Fairfield, Ohio).
  • Poly(ethylene glycol) 2000 monomethyl ether (MePEG 2000 ) and polyethylene glycol) 2000 (diOH-PEG 2000 ) were purchased from Fluka (Buchs, Switzerland). These materials were used without further purification. All solvents were analytical grade.
  • the PLA-PEG diblock copolymer was synthesized through the polycondensation of lactic acid on MePEG2000, in the absence of any catalyst. Briefly, 10 g of MePEG 2000 and 10 g of an aqueous lactic acid solution were placed in a round-bottomed bottle. We performed the polymerization by distilling out water from lactic acid at 140° C. for 24 hr using a system composed of a Rotavapor® R-210 (Buchi Labortechnik AG, Switzerland) and vacuum pump V-700 (Buchi Labortechnik AG). The products were recovered by precipitation in an excessive amount of ethanol.
  • the PLA-PEG-PLA triblock copolymer was synthesized according to the same procedure with diOH-PEG 2000 used instead of MePEG 2000 . The product was recovered by precipitation in cold ethanol ( ⁇ 10° C.), and the yield was about 30 wt %.
  • MALDI-TOF MS was recorded on a Waters® MALDI micro MXTM mass spectrometer (Milford, Mass.) equipped with a nitrogen laser (337 nm). All spectra were recorded in the reflection mode with an acceleration voltage of 12 kV.
  • the irradiation targets were prepared from 0.1% trifluoroacetic acid (Riedel-de Haen, Seelze, Germany) in an acetonitrile/water mixture at a ratio of 50/50 (v/v) with ⁇ -cyano-4-hydroxy cinnamic acid (Sigma, Steinheim, Germany) as the matrix and sodium trifluoroacetate (Na-TFA, Fluka) as the dopant.
  • sample solutions were then spotted on a MALDI sample plate and air-dried before analysis.
  • GPC was performed with a setup composed of an isocratic pump (Waters® high-performance liquid chromatography (HPLC) Model 510), a refractive index detector (Waters®410 differential refractometer), and two columns connected in series, one PLgel 5- ⁇ m mixed-C column (100- ⁇ pore size, 7.5 ⁇ 300 mm, Polymer Laboratories, Ltd., Shropshire, United Kingdom), and one PLgel 3- ⁇ m column (100- ⁇ pore size, 7.5 ⁇ 300 mm).
  • HPLC high-performance liquid chromatography
  • Waters®410 differential refractometer Waters®410 differential refractometer
  • the mobile phase was tetrahydrofuran and the flow rate was 0.8 mL/min, Data were expressed with respect to polystyrene standards (Polysciences, Inc., Warrington, Pa.).
  • 1 H-NMR spectra were recorded at room temperature with a Varian VXR 300-MHz spectrometer (Varian, Palo Alto) with dimethyl sulfoxide-4 (Aldrich, Steinheim, Germany) and tetramethylsilane as the solvent and shift reference, respectively.
  • the polymer aqueous solution [120 mg of polymer dissolved in 0.8 mL of phosphate-buffered saline (PBS)] and 1.1 mL of squalene oil (Sigma, Steinheim, Germany) were emulsified with a Polytron® PT 3100 homogeniser (Kinematica AG, Lucerne, Switzerland) under 6000 rpm for 5 min.
  • the emulsified formulations served as stocks for further physicochemical characterizations.
  • the stability test was performed by placing each formulation at 4 and 37° C. and observed the visual aspects.
  • the stock emulsion was redispersed in the PBS before the measurement by using the laser light-scattering technique with a Brookhaven 90 plus particle size analyzer (Brookhaven Instruments Limited, New York).
  • In vitro release experiments were performed with the inverted dialysis tube method.
  • Formulations containing OVA albumin from chicken egg white, Grade V, Sigma; 3 mg/0.3 mL
  • were placed in a dialysis chamber (cutoff 0.2 ⁇ m, Pall Life Sciences, Ann Arbor, Mich.).
  • the device was immersed in a 50-mL centrifuge tube containing 2 mL of PBS and left to stand at 37° C. At different time intervals, 100 ⁇ L of sample were aspirated from the medium outside of the chamber and replaced with 100 ⁇ L of fresh PBS buffer. The OVA release was regularly determined by the bicinchinonic acid method (BCATM protein assay kit, Pierce, Rockford, Ill.).
  • FIG. 4 shows the synthesis and the chemical structure of the block copolymers.
  • the PLA-PEG diblock copolymer was synthesized by the polycondensation of lactic acid in the presence of monomethoxy PEG, which resulted in a copolymer composed of a hydrophilic block PEG and a lipophilic block PLA.
  • the triblock copolymer PLA-PEG-PLA was obtained from the polymerization of lactic acid in the presence of dihydroxyl PEG.
  • PLA compounds are synthesized by the ring-opening polymerization of lactide (a cyclic diester of lactic acid) or the polycondensation of lactic acid. Although the latter is a reasonably low-cost and straightforward method for synthesizing polymers bearing PLA segments this route generally leads to oligomers with low-molar-mass chains.
  • Table 2 The molecular characteristics of the resulting copolymers are summarized in Table 2.
  • FIGS. 5 a - 5 b show the MALDI-TOF MS spectra of PLA-PEG and the corresponding MePEG 2000 .
  • the subsidiary peaks were assigned to the isotopes of elements.
  • OE oxyethylene units
  • LA lactyl
  • the major five polymer species between 2030 and 2100 m/z in the spectra of PLA-PEG ( FIG. 5 b ) are represented as follows:
  • HLB hydrophilic-lipophilic balance
  • HLB PLA/PEG 20 x ( W PEG /W PLA-PEG )
  • W PEG /W PLA/PEG is the weight ratio of the hydrophilic portion of the main-chain polymer and was obtained from Mn PEG /Mn PLA-PEG .
  • the most lipophilic portion has an HLB number approaching 0, and the most hydrophilic portion has a number of about 20.
  • GPC is a separation technique based on the molecular hydrodynamic volume. By comparing with a standard curve of a known MW species, the relative MW of the samples could be easily calculated.
  • Table 2 shows molecular characteristics of the block copolymers of PEG and lactic acid initiated by PEG. The average MW increased after the introduction of lactic acid chains onto the prepolymer PEG.
  • the GPC traces of PLA-PEG and PLA-PEG-ALA exhibited monomodal distributions and reflected rather narrow MW distributions, which indicated the absence of residual low-molecular-weight species. 1 H-NMR data revealed that these low-molecular-weight species consisted of unreacted lactic acid and/or LA-rich species.
  • the LA units/OE units molar ratio or [LA]/[OE] was determined from the integrations of the proton resonances due to PEG blocks at 3.6 ppm and to PLA blocks at 1.5 ppm on the 1 H-NMR spectra.
  • the single peak at 3.3 ppm assigned to the hydrogens of methyl groups was also detected on the NMR spectra of MePEG 2000 and PLA-PEG.
  • the MW of the copolymers was determined according to the following relationship:
  • PLA-PEG and PLA-PEG-PLA could be used as emulsifiers
  • the polymer aqueous solution was homogenized with squalene oil, which resulted in an isotropic emulsified formulation.
  • the emulsions remained stable for a few weeks when they were stored at 4° C. After 2 weeks, 5% of water disassociated, but beyond this no further water disassociation from the emulsion occurred.
  • the isotropic emulsion could be reformed by vortex mixing. Little difference was observed between the PLA-PEG- and PLA-PEG-PLA-stabilized emulsions.
  • the size distribution of the emulsions and in vitro OVA release were measured to identify the dispersion characteristics of the resulted emulsions and to understand the effect of the copolymer in the emulsification process.
  • the emulsion was redispersed in the PBS and the size distribution was measured with a particle size analyser.
  • a water-in-oil (W/O) emulsion droplet remains floating on a water surface, and the particle size is undetectable with a light-scattering technology.
  • W/O water-in-oil
  • an O/W emulsion droplet can stand only for seconds in an aqueous phase and then diffuses into the water.
  • the dynamic light-scattering pattern showed that PLA-PEG or PLA-PEG-PLA was a suitable emulsifier for squalene/water emulsions and yielded narrowly distributed nanoparticles in the PBS.
  • Table 3 shows physicochemical characteristics of the squalene emulsions based on PLA-PEG and PLA-PEG-PLA.
  • 7 shows the cumulative release of OVA from different formulations. Initially, a fast release was observed in the nonformulated OVA, from which more than 80% of loaded OVA were released into the outside PBS medium within the first 50 hr.
  • the PLA-PEG/squalene or PLA-PEG-PLA/squalene emulsion allowed a slight delay, then the protein was quickly released.
  • the visual aspect showed that the emulsions remained stable only 5% of water disassociated at the bottom over 200 h at 37° C.
  • Surfactants as emulsifiers can be defined by their HLB values, which give infbrmation about the relative affinity to aqueous and oily phases.
  • a lipophilic emulsifier renders a W/O emulsion with a high affinity to an oily phase, whereas a hydrophilic emulsifier renders an O/W emulsion with a high affinity to an aqueous phase.
  • degradable aliphatic polyesters used for vaccine or protein delivery have been in the form of injectable microspheres or implant systems.
  • Such systems require complicated fabrication processes using organic solvents, which may cause denaturation when antigens (virus or proteins) are to be encapsulated.
  • the systems require polymers with high MW (generally >50,000 Da), which require severe polymerization conditions (extreme temperature and pressure and toxic catalysts).
  • the stable squalene; water emulsions were obtained with PEG-containing PLA oligomers as emulsifiers without the addition of any other stabilizer.
  • the bioactive candidates could be either surface attached to or encapsulated within a core oil.
  • the obtained emulsions had a high affinity to water so that nanoparticles were obtained after they were redispersed into the PBS. Moreover, no catalyst was required for the preparation of the designed polymers.
  • the emulsified formulation developed here was free of organic solvents.
  • PLA/PEG diblock and triblock copolymers with high HLB values were synthesized by the direct polycondensation of an aqueous lactic acid solution on monomethoxy PEG or dihydroxyl PEG in the absence of a catalyst.
  • MALDI-TOF MS data allowed us to calculate the repeat unit masses and end-group masses so that the molecular structure between the diblock and triblock copolymers could be distinguished.
  • the obtained copolymers could serve as hydrophilic emulsifiers and rendered stable O/W emulsified nanoparticles when the polymer aqueous solution was homogenized with squalene oil.
  • This example illustrates the use of an amphiphilic polymer, namely, poly(ethylene glycol)-block-poly(lactide-co- ⁇ -caprolactone) (PEG-b-PLACL), as an emulsification agent to render different types of vaccine formulations.
  • the hydrophilic block was made of PEG because of its availability, water-solubility, and high biocompatibility.
  • Degradable aliphatic polyesters in particular polylactides (PLA) and poly( ⁇ -caprolactone) (PCL), have been widely used as medical and drug delivery devices with FDA approval. PLA with variable chain stereoregularity provides a worthwhile means to adjust the rate of degradation, in addition to its physical and mechanical properties.
  • the degradation products of PCL have a relatively higher pKa than those of poly(lactide-co-glycolide) (PLG) (4.8 for ⁇ -hydroxycaproic acid, and 3.8 for lactic acid and glycolic acid at 25° C.), and they may provide more conservation of protein molecular integrity when being used for a long-term controlled delivery of proteins.
  • PLACL was thus chosen as a lipophilic block for its fast degradation characteristics.
  • its amorphous nature provides good affinity between the polymer matrix and oil solutions.
  • Squalene was selected as the core oil because unlike mineral oil., it is natural and metabolizable.
  • the excipient use of Span®85 in the oily phase is also positively indicated, as it is an emulsifying agent in licensed human vaccines.
  • PEG-b-PLACL was synthesized by the ring-opening polymerization of lactide (Aldrich) and ⁇ -caprolactone (CL. Aldrich), using SnOct 2 (stannous octoate, Sigma) as a catalyst and MePEG (polyethylene glycol 5000 monomethyl ether, Fluka) as an initiator. Briefly, predetermined amounts of MePEG (2.1 g), lactide (0.58 g), and ⁇ -caprolactone (0.47 g) were placed in a dried round-bottomed bottle, and the appropriate amount of SnOct 2 (30 mg) was added as a solution in dried toluene (10 mL). The polymerization was performed at 140° C.
  • the resulted polymer was characterized by 1 H nuclear magnetic resonance ( 1 H NMR) and gel permeation chromatography (GPC). 1 H NMR spectra were recorded at room temperature with a Varian VXR 300 MHz spectrometer (Varian, Palo Alto) using deuterated chloroform as a solvent.
  • the molar ratio of oxyethylene to lactyl units to caproyl units or [OE]:[LA]:[CL] was determined from the integrations of the proton resonances due to PEG blocks at 3.6 ppm, to PLA blocks at 5.2 ppm and to PCL blocks at 4.1 ppm on the NMR spectra.
  • M nPEG-b-PLACL 5000+72 ⁇ 50000/44 ⁇ [LA]/[OE]+ 114 ⁇ 5000/44 ⁇ [ CL]/[OE]
  • HLB hydrophilic-lipophilic balance
  • HLB PEG-b-PLACL 20 ( W PEG /W PEG-PLACL )
  • W PEG /W PEG-b-PLACL is the weight ratio of the hydrophilic portion of the main chain polymer and is obtained from M n PEG / M n PEG-b-PLACL .
  • GPC was performed by using a setting composed of a Waters 510 HPLC pump, a Waters 410 differential refractometer, one PLgel mixed-C 5 ⁇ m 100 ⁇ column (7.5 ⁇ 300 mm), and one PLgel 3 ⁇ m 100 ⁇ column (7.5 ⁇ 300 mm), and the mobile phase being THF and the flow rate being 0.8 mL/min. Data were expressed with respect to polystyrene standards from Polysciences. A unimodal and narrow molecular weight distribution (polydispersity index being 1.1) was observed in GPC chromatograms of PEG-b-PLACL and the corresponding MePEG 5000 , which indicated a full initiation of the macroinitiator.
  • the fact that the polymer PEG-b-PLACL aqueous solution forms micelles is an indication that the polymer possesses an amphiphilic nature. This was confirmed by dye solubility experiments and light scattering analysis. Briefly, five milligrams of polymer were added to 1 mL of PBS in the presence of the water-insoluble dye diphenylhexatriene (DPH, Sigma), which is known to dissolve in the hydrophobic core of polymeric micelles or aggregates. After sonication and centrifugation, an abrupt enhancement in the ultraviolet (356 nm) absorption of the dye was observed, which was an indication, of micelle formation. The particle size distribution was determined by using the laser light scattering technique with a Brookhaven 90 plus particle sizer (Brookhaven Instruments Limited).
  • emulsified formulations serving as stocks for further physicochemical characterizations, including stability, the droplet test, microscopic aspects, and in vitro release.
  • the stability test was performed by placing each formulation at 4° C. and observed the emulsion at predetermined time points (2 weeks, 1 month, 3 months, 6 months, 1 year).
  • the droplet test was assessed by placing a droplet (20 ⁇ L) of emulsion into a water-containing beaker (200 mL).
  • the microscopic aspects of the emulsions were investigated by redispersing them (100 ⁇ L) into a continuous phase (900 ⁇ L) and monitoring with an Olympus DP70 microscope. Particle size distribution was determined by using the laser light scattering technique.
  • OVA-containing formulations (3 mg per 0.3 mL) were placed in a dialysis chamber (cutoff 0.2 ⁇ m) and the device was then immersed in a 50 mL centrifuge tube containing 2-mL of ‘PBS at 37° C. At different time points, 100 ⁇ L of sample were aspirated from the medium outside of the chamber and replaced with 100 ⁇ L of fresh PBS buffer.
  • the OVA release was regularly determined by the bicinchinonic acid method (BCATM protein assay kit, Pierce).
  • mice Five-week old female BALBk mice were obtained from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) and acclimatized for at least 1 week at the animal facility of the National Health Research Institutes (NHRI, Miaoli, Taiwan) before use. All animal studies were approved by the Animal Committee of the NHRI. Mice were immunized subcutaneously with syringe needles of 27G ⁇ 1 ⁇ 2′′ at weeks 0, 2, and 4 by 0.5 ⁇ g of OVA in PBS or formulated with PEG-b-PLACL/squalene or PEG-b-PLACL/squalene/Span®85 or aluminum phosphate suspension (alum, 150 ⁇ g per dose).
  • the PEG-b-PLACL/squalene and PEG-b-PLACL/squalene/Span®85 formulations were prepared by redispersing 100 ⁇ L of a stock emulsion (see MATERIALS AND METHODS: Polymer-Based Emulsions) into 900 ⁇ L of PBS before the injections, which resulted in the formulations with ⁇ 5% oil. Sera and spleen collections were performed to determine B- and T-cell responses, respectively.
  • mice were bled at the lateral tail vein and the collected sera were stored at ⁇ 30° C. until assaying.
  • the presence of OVA-specific antibodies in the sera was determined by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • 100 ⁇ L of diluted OVA (10 ⁇ g/mL) were coated onto 96-well microtiter plates with 0.05M carbonate buffer (pH 9.6) for overnight incubation at 4° C.
  • the coated plates were washed twice with PBS containing 0.05% Tween®20 (Sigma) and then blocked with 5% nonfat milk in PBS at room temperature for 2 h.
  • the mouse spleen was removed aseptically and placed in an eppendorf containing 1 mL of culture medium (cRPMI) consisting of RPMI 1640 (SAFC, Kansas) with 2 mM L-glutamine, and supplemented with 25 mM HEPES (Gibco, Invitrogen, NY), 0.05 mM 2-mercaptoethanol, 10% heatinactivated fetal bovine serum (FBS, HyClone, Perbio) and 1% antibiotics.
  • cRPMI culture medium
  • RPMI 1640 SAFC, Kansas
  • HEPES Gibco, Invitrogen, NY
  • FBS heatinactivated fetal bovine serum
  • FBS heatinactivated fetal bovine serum
  • the cell suspension was collected in a 50 mL centrifuge tube and then centrifuged at 1000 rpm for 5 min. To remove the erythrocytes, the cell pellet was resuspended in 5 mL of the ACK lysis buffer placed to room temperature for 1 min, terminated the reaction with 20 mL RPMI 1640, followed by centrifugation for 5 min. The pellet was washed twice with cRPMI and resuspended in 5-mL of cRPMI. After cell counting with a hemocytometer using the trypan blue dye exclusion technique, U-bottomed 96-well plates were seeded with 2 ⁇ 10 5 cells in cRPMI at a total volume of 200 ⁇ L per well.
  • a diblock copolymer consisting of 75 wt % of the hydrophilic block PEG and 25 wt % of the lipophilic block PLACL with molecular weight of 7000 daltons, polydispersity index of 1.1 and a calculated HLB of 15 was synthesized via the ring-opening polymerization of lactide and ⁇ -caprolactone on monomethoxy PEG with the catalyst SnOct 2 .
  • the amphiphilic nature of PEG-b-PLACL was confirmed by self-association of micelles in the polymer aqueous solution. Dynamic light scattering displayed polymeric micelles with a unimodal size distribution with an average diameter of 18.2 ⁇ 0.4 nm. Preliminary immunogenicity studies however showed that the polymeric aqueous solution had no adjuvant effect because they induced the same level of antigen (OVA)-specific antibodies as those formulations without the adjuvant (data not shown).
  • OVA antigen
  • the amphiphilic polymer was used as an emulsifier to make different emulsion-type formulations by homogenizing a mixture of the polymer aqueous solution and squalene or the squalene/Span®85 oil ( FIG. 8 a ).
  • Table 4 lists the physicochemical characteristics of various formulations based on the bioresorbable polymer PEG-b-PLACL and selected oils.
  • the emulsified formulation was white and isotropic from the top to the bottom ( FIG. 8 b ).
  • FIG. 8 c shows the stability of the emulsions stored at 4′′C.
  • the emulsion PEG-b-PLACL/squalene/Span®85 was stable for at least 1 year without occurrence of phase separation. Conversely, ⁇ 10% of free oil at the surface layer disassociated from the emulsion PBS/squalene/Span®85 after I month. In the case of the emulsion PEG-b-PLACL/squalene with aqueous/oily being 5/5 w/w, 20% of water disassociated under the same storage conditions after 2 weeks, but beyond this no further water disassociation from the emulsion occurred. An isotropic emulsion could be reformed by homogenization in the same condition or simply by vortex mixing.
  • the type of dispersion of the emulsion was examined by a droplet test, microscopic aspects, and in vitro release.
  • the droplet test allowed the identification of a continuous phase of emulsion.
  • a droplet of PBS/squalene/Span®85 emulsion remained floating on the water surface after 24 h ( FIG. 9 a ) even after gentle hand stirring of the beaker.
  • the PEG-b-PLACL/squalene emulsion droplet could stand for only one half-hour in an aqueous phase and then diffused into the water ( FIG. 9 b ), a feature similar to the PEG-b-PLACL/squalene/Span®85 system.
  • the emulsion drops were invisible under an optical microscope since they were crowded in the dispersed phase.
  • the size distribution of the emulsion was investigated by redispersing the emulsion in the continuous phase and measuring the size with a microscope and particle sizer.
  • the emulsion PEG-b-PLACL/squalene was composed of nonhomogeneous particles with a bimodal distribution. Two different sizes of particles were observed, relatively large particles of about 10 ⁇ m and smaller particles of about 1 ⁇ m. By contrast, homogeneous fine particles less than 1 ⁇ m were observed with an optical microscope when redispersing the PEG-b-PLACL/squalene/Span®85 in the PBS ( FIG. 10 b ).
  • the dynamic light scattering pattern showed a unimodal distribution with an average diameter of 457.7 ⁇ 25.8 nm.
  • Stabilized particles of ⁇ 1-10 ⁇ m in diameter are appropriate for uptake by antigen-presenting cells (APCs) to facilitate the induction of potent immune responses due to the pseudo-natural targeting of antigens.
  • FIG. 11 shows the cumulative release of OVA from various formulations: (-x-) no adjuvant, (open circle) the aqueous solution PEG-b-PLACL, (open square) the O/W emulsion PEG-b-PLACL/Squalene, (open triangle) the W/O/W emulsion PEG-b-PLACIL/squalene/Span®5, (filled circle) the W/O emulsion PBS/squalene/Span®85.
  • a fast release was observed in the case of OVA without adjuvant from which more than 80% of loaded OVA were released into the outside PBS medium within the first 50 h.
  • the polymer aqueous solution or PEG-b-PLACL/squalene emulsion allowed a slight delay but the protein was quickly released.
  • the emulsion PEG-b-PLACL/squalene/Span®85 released less than 50% of OVA during the same period of time. Afterwards, the protein release increased continuously until it reached an equilibrium concentration in the inside and outside of the dialysis device.
  • the PBS/squalene/Span®85 emulsion presented a well depot effect on OVA so that hydrophilic OVA was slowly released over 200 h.
  • FIG. 11 b shows the recovered formulations after 200 h experiments. Phase separation occurred in the formulation PBS/squalene/Span®85. By contrast, the PEG-b-PLACL-based emulsion remained stable with clear layers' of water at the bottom. The protein (ovalbumin as an example) initially encapsulated within the emulsion was almost released at this time, which indicated that the hydrophilic bioactive agents (or antigens) trapped within the polymer-emulsified oily emulsion were released from the core oil to the surface mostly by diffusion, but also to a lesser extent by degradation mechanisms and emulsion breaks.
  • the hydrophilic bioactive agents or antigens
  • the serum antibody IgG titers as well as isotypes IgG1 and IgG2a were significantly enhanced for the group of PEG-b-PLACL/squalene or PEG-b-PLACL/squalene/Span®85- or alum-formulated OVA in comparison to the free OVA group (p ⁇ 0.05).
  • the ratio between the geometric mean titer (GMT) obtained with PEG-b- PLACL/squalene/Span®85-formulated OVA vaccine and the GMT obtained with vaccine alone was found to be 19.9 at week 4, in comparison with the group of PEG-b-PLACL/squalene being 2.4 and the group of alum being 6.3.
  • PEG-b-PLACL/squalene-formulated OVA induced comparable levels of serum antibody titers as alum-formulated OVA within 10 weeks.
  • the highest antibody responses were elicited in the group of PEG-b-PLACL/squalene/Span®85-formulated OVA, in which statistical significance with respect to alum was detected at weeks 2 and 4, that is, in the early stages after immunization.
  • the immunogenicity increase was probably due to appreciable particle size and/or carrier/depot activity.
  • Sera were collected from blood and the antibody titers were measured by ELISA. The data are presented as geometric mean titers with 95% confidence intervals of five mice per group.
  • a P ⁇ 0.05 Comparison with free OVA group at the same time point.
  • b P ⁇ 0.05 Comparison with alum-formulated OVA group at the same time point.
  • ⁇ 50 means undetectable in an initial dilution of 1:50.
  • FIG. 12 a showed that following one immunization, OVA alone did not induce antigen-specific proliferative response well due to the low dosage of antigen so that the stimulation index of the OVA/PBS group was only slight higher than the threshold value.
  • T helper type 1 cytokine a predominant T helper type 1 cytokine, was detected in the supernatants of splenocyte collected from mice treated with PEG-b-PLACL/squalene- and PEG-b-PLACL/squalene/Span®85- formulated OVA, following in vitro restimulation of splenocytes with OVA.
  • T helper type 2 cytokine IL-4 was measured at the same or reduced level as OVA alone.
  • HLB hydrophilic-lipophilic balance
  • the emulsion PEG-b-PLACL/squalene/Span®85 is composed of two emulsifiers and renders a water-in-oil-in-water (W/O/W) multiphase emulsion ( FIG. 13 ).
  • W/O/W water-in-oil-in-water
  • FIG. 13 From a viewpoint of emulsion stability, oil droplets with a small particle size and homogeneous distribution are more stable, which are however strongly influenced by the optimization of the surfactant system and the emulsification process.
  • Homogenization of PEG-b-PLACL-containing aqueous solution and the Span®85-contained oily phase provides a potential way of stabilizing emulsified particles both at storage and at postinjection stage conditions.
  • a polymer can play different roles in a particulate delivery system. The most direct role is to provide a matrix or a vehicle that builds the microparticle.
  • Degradable PLG, PLA, and PCL used for vaccine or protein delivery have mostly been in the form of injectable microspheres or implant systems. Such systems require complicated fabrication processes using organic solvents and may cause denaturation when antigens (virus or proteins) are to be encapsulated.
  • the systems require polymers with high molecular weight (generally >100,000 Da), which require severe polymerization conditions (extreme temperature and pressure and toxic catalysts).
  • amphiphilic copolymers such as poly (ethylene glycol)-block-poly(propylene sulphide)-blockpoly(ethylene glycol) and poly(ethylene glycol)-block-polyoxypropylene-block-poly(ethylene glycol) (known as Pluronic® or Poloxamers) could be formed as polymersomes, as oil free thermosensitive hydrogels or as a surfactant to render an emulsion (known as TiterMax®) in vaccine delivery systems.
  • Pluronic® or Poloxamers poly(ethylene glycol)-block-polyoxypropylene-block-poly(ethylene glycol)
  • the emulsified vaccine delivery systems have several advantages over traditional vaccine adjuvants. Firstly, synthetic polymeric emulsifier is reproducible from batch to batch, and the relative hydrophobic/hydrophilic balance can be easily manipulated by the amounts of monomer used, thus producing a broad range of emulsifier characteristics. Secondly, unlike antigen adsorption onto alum, the system allows either the surface attachment or encapsulation of antigens, and the emulsified formulation can be stored at or below room temperature as a stock before injection.
  • the acidic condition is not required in the preparation of PEG-b-PLACL and/or Span®85 emulsified delivery systems because both are nonionic emulsifiers.
  • bioresorbable polymeric emulsifiers with a hydrophobic block that is degradable, show bulk degradation and further resorb in vivo.
  • the raw materials for polymer synthesis described here are commercially available and frequently used for temporary therapeutic applications.
  • the formulation is easy for preparation, that is, no complicating processes or supplemental equipment are required and thus the cost is reduced.
  • the polymer-emulsified delivery system is free of organic solvents, in contrast to the common polymeric microspheres.
  • bioresorbable diblock tri-component copolymer PEG-b-PLACL as an emulsifier and rendered an O/W or W/O/W multiphase emulsion when the polymer aqueous solution was homogenized with squalene or the squalene/Span®85 mixture.
  • Novel polymer-emulsified formulations have high affinity to water so that the stock OVA-containing emulsion could be redispersed into PBS before injection, thus resulting in fluid emulsion (only 5% of oil within the emulsion) with homogeneous particles rangimi between 1 and 10 ⁇ m.
  • the emulsified particles could serve as either carriers or vehicles to deliver biologically active agents (ovalbumin as an example) to APCs in a targeted and prolonged manner, thus effectively enhancing immunity.
  • biologically active agents ovalbumin as an example
  • These formulation have potential to be used in adjuvants for prophylactic and therapeutic vaccine candidates.
  • Such applications include single-dose multivalent vaccine development and via alternative immunization routes, such as intramuscular or transdermal administration.
  • PEG-b-PLACL bioresorbable diblock tri-component co-polymer polyethylene glycol)-block-poly(lactide-co- ⁇ -caprolactone)
  • PELC water-in-oil-in-water multiphase emulsion-type vaccine delivery system
  • PEG-b-PLACL served as a hydrophilic emulsifier
  • Span®85 acted as a hydrophobic emulsifier to stabilize the water/squalene interface, resulting in a stable and homogeneous nanoemulsion.
  • HA hemagglutinin
  • inactivated virus induced more potent antigen-specific antibodies, hemagglutination inhibition, and virus neutralization than HA (5 ⁇ g) of non-adjuvanted virus, demonstrating the antigen economization of the PELC-based vaccine.
  • T-cell proliferative responses as well as IFN- ⁇ and interleukin-4 (IL-4) secretion were significantly enhanced after formulation with the PELC emulsion.
  • the vaccine used in this study was the formalin-inactivated whole-virus vaccine NIBRG-14, which was kindly supplied by the UK National Institute of Biological Standard and Control, NIBSC.
  • the vaccine was derived from a reassorted H5N1 vaccine strain containing modified HA and neuraminidase (NA) from a highly pathogenic avian influenza strain A/Vietnam/1194/2004 and propagated in Madine-Darby canine kidney (MDCK) cells.
  • Formalin-inactivated vaccines were prepared with 0.1% formalin at 37° C. for 24 h.
  • the HA content was determined by single radial diffusion (SRD). Production details for the H5N 1 vaccine candidate are reported elsewhere.
  • the diblock co-polymer PEG-b-PLACL was synthesized by the ring-opening polymerization of lactide and ⁇ -caprolactone on monomethoxy PEG as previously described.
  • the PEG-b-PLACL consisted of 75 wt % hydrophilic block PEG and 25 wt % lipophilic block PLACL.
  • the calculated hydrophilic lipophilic balance (HLB) value was 15 with a molecular weight of 7000.
  • PELC is an emulsion-type vaccine delivery system based on PEG-b-PLACL, Span®85, and squalene.
  • a PEG-b-PLACL-containing aqueous solution 120 mg dissolved in 0.8 mL of phosphate-buffered saline (PBS)
  • PBS phosphate-buffered saline
  • an oily phase containing a squalene/Span®85 mixture 85/15 v/v
  • the emulsified formulation was stored at 4° C. until use.
  • the PELC-adjuvanted vaccine was formulated by re-dispersing 200 ⁇ L of the stock PELC emulsion in 1800 ⁇ L of bulk vaccine before injection.
  • the size distribution of the emulsion droplets was determined with a microscope (Olympus DP70) and the laser light scattering technique (Brookhaven 90 plus particle size analyzer, Brookhaven Instruments Limited).
  • mice Five-week-old female BALB/c mice were obtained from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) and acclimatized for at least one week at the animal facility of the National Health Research Institutes (NHRI, Miaoli, Taiwan) prior to use.
  • NHRI National Health Research Institutes
  • H5N1 influenza vaccine All mice were vaccinated intramuscularly (i.m.) with one of two different closes (0.5 ⁇ g or 5 ⁇ g HA) administrated with or without PELC. Serum and tissue collection were performed to determine B- and T-cell responses. Serum samples were collected from immunized mice and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) as well as by hemagglutination inhibition titration and viral neutralizing assays.
  • ELISA enzyme-linked immunosorbent assay
  • NIBRG-14-specific antibodies in the sera was determined via ELISA. Briefly, 96-well microtiter plates were coated with 100 mL of dilute inactivated virus (1 ⁇ g/mL) and incubated overnight at room temperature. The coated plates were washed once with PBS containing 0.05% Tween®20 (Sigma) and blocked with 1% bovine serum albumin (BSA, Sigma) in PBS at room temperature for 2 h. Diluted sera (starting dilution 1:1000, serial two-fold serum dilutions) from immunized mice were applied to the blocked wells at room temperature for 2 h.
  • BSA bovine serum albumin
  • HRP-conjugated goat anti-mouse IgG (ICN Cappel, 1:5000) was added to all wells at room temperature for 30 min.
  • the assay was developed using the substrate solution 2, 20-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS® Peroxidase, KPL) for 20 min at room temperature and shielded from light. Plates were read at 405 nm with an ELISA plate reader (Thermo Multiskan® spectrophotometer, Vantaa, Finland).
  • the HI test was based on the ability of the specific anti-influenza antibodies to inhibit hemagglutination of turkey red blood cells (RBCs) by influenza virus HA.
  • Non-specific inhibitors of agglutination were removed by heat treatment and addition of receptor-destroying enzymes.
  • serum samples (two-fold dilutions starting at an initial dilution of 1:10) were incubated with four hemagglutination units of influenza strain. Turkey RBCs were then added and agglutination inhibition was scored.
  • the serum titer was expressed as the reciprocal of the highest dilution that demonstrated complete HI.
  • the seroprotection rate (SPR, %) was calculated from the proportion of mice achieving a post-vaccination titer ⁇ 40.
  • VN Virus Neutralization
  • the NIBRG-14 virus in 200 TCID50 (50% tissue culture infective dose) per well was incubated with two-fold-diluted mice sera at a starting dilution of 1:40. The mixtures of virus and serum were transferred to monolayers of MDCK cells and incubated at 37° C. and 5% CO 2 for 4 days.
  • the neutralizing titer was expressed as the reciprocal of the highest serum dilution at which the infectivity of the H5N1 virus 200 TOD50 for MDCK cells was completely neutralized in 50% of the wells. Infectivity was identified by the presence of cytopathy on Day 4 and the titer was calculated using the ReedeMuench method.
  • the statistical significance was determined by performing a two-tailed Student's t-test on log-transformed values.
  • Cell suspensions were prepared by mashing the spleen through a cell strainer with a syringe plunger. The resulted suspension was collected in a 50-mL tube and centrifuged at 1000 rpm for 5 min. To remove erythrocytes, the cell pellet was resuspended in 5 mL of RBC lysis buffer (150 mM NH 4 Cl, 1 mM KHCO 3 and 0.1 mM EDTA (pH 7.3), Biolegend, CA) and incubated at room temperature for 1 min.
  • RBC lysis buffer 150 mM NH 4 Cl, 1 mM KHCO 3 and 0.1 mM EDTA (pH 7.3), Biolegend, CA
  • reaction was then terminated with 20 mL of RPMI-1640 and the mixture was centrifuged for 5 min, The pellet was washed twice with cRPMI and resuspended in 5 mL of cRPMI. After cell counting with a hemacytometer by the trypan blue dye exclusion, U-bottomed 96-well plates were seeded with 2 ⁇ 10 5 cells in cRPMI at a total volume of 200 ⁇ L per well. Cells were stimulated in triplicate in the presence or absence of 2.5 ⁇ g HA/mL of inactivated NIBRG-14 virus. Concanavalin A (Con A, 5 ⁇ g/mL, Sigma) was used to induce a maximal proliferative response.
  • Concanavalin A Con A, 5 ⁇ g/mL, Sigma
  • the PELC-formulated influenza candidate vaccine consisted of an inactivated virion and a pre-emulsified PELC stock (see Materials and methods:Adjuvant preparation). Prior to the injection, the PELC stock and inactivated virions were mixed to form homogeneous particles. The size distribution of the particles ranged from 200 to 400 nm in diameter ( FIG. 14 ), which was consistent with the definition of nanoemulsion. Importantly, it has been reported that this nanoseale dimension is conducive to uptake by antigen-presenting cells, which facilitates the induction of potent immune responses.
  • BALB/c mice were intramuscularly immunized with 0.5 ⁇ g or 5 ⁇ g HA of inactivated NIBRG-14 virus formulated with or without PELC.
  • Table 6 shows NIBRG-14-specific IgG1, and IgG2a antibodies elicited in BALB/c mice following a single intramuscular dose of H5N1 inactivated virus vaccine. The elicited antigen-specific antibodies are shown in Table 6.
  • *P ⁇ 0.05 comparison with the group of 0.5 ⁇ g HA without adjuvant at the same time point.
  • # P ⁇ 0.05 comparison with the group of 5 ⁇ g HA without adjuvant at the same time point.
  • ⁇ 1000 means undetectable in an initial dilution of 1:1000.
  • a Titrations do not reflect absolute concentrations as can be seen in the IgG2a subtype assays that are more sensitive than the total IgG assays.
  • the HI activity assay is the most common way to determine the efficacy of an influenza vaccine.
  • Table 7 shows that HI antibody responses were elicited in BALB/c mice following a single intramuscular dose of inactivated H5N1 virus vaccine formulated with or without PELC.
  • sera from the mice vaccinated with 0.5 ⁇ g HA of non-adjuvanted inactivated virus elicited an HI geometric mean titer (GMT) of 6 and 9 at Weeks 2 and 4, respectively.
  • the highest GMT responses were 59 at Week 12 and 33 at Week 26.
  • the HI titer was slightly enhanced at Weeks 2, 4, and 8. There were however no statistically significant differences (Pz) in HI titers between the 0.5 ⁇ g and 5 ⁇ g HA groups.
  • the PELC-adjuvanted vaccines were capable of inducing higher HI titers than those developed from the virus alone (P ⁇ 0.05).
  • the HI titer was significantly higher than that induced by non-adjuvanted, 5 ⁇ g HA of inactivated virus.
  • the seroprotection rate (SPR, %) is the percentage of mice achieving a post-vaccination titer ⁇ 40.
  • SPR The seroprotection rate
  • *P ⁇ 0.05 comparison with the group of 0.5 ⁇ g HA without adjuvant at the same time point.
  • # P ⁇ 0.05 comparison with the group of 5 ⁇ g HA without adjuvant at the same time point.
  • ⁇ 10 means undetectable in an initial dilution of 1:10.
  • VN Virus Neutralization
  • VN assays were performed to provide a more functional measure of vaccine-induced immunity. As shown in Table 8, the neutralizing antibody titers were slightly enhanced When the amount of non-adjuvanted antigen administered was increased. In contrast, when the vaccine was formulated with PELC, the neutralizing antibody titers were dramatically enhanced. The highest neutralizing antibody titers were induced by the PELC-adjuvanted 5 ⁇ g HA group.
  • the VN capability of PELC-formulated, inactivated virus was complementary to its adjuvanticity demonstrated by HI titers. Thus, a combination of PELC and inactivated virus may induce sufficient and sustainable protective antibodies.
  • inactivated virus could induce higher antibody titers, higher HI activity, and higher VN activity than 5 ⁇ g HA of inactivated virus alone.
  • FIG. 1 SA shows that following one vaccination, the virus alone did not induce a notable antigen-specific proliferative response.
  • SI Stimulation Index
  • IFN- ⁇ is a predominant T helper type 1 (Th1) cytokine relevant to virus-specific cytotoxic T lymphocyte (CTL) activity
  • IL-4 is a common T helper type 2 (Th2) cytokine. Therefore, an antigen formulated with PELC may not only increase the humoral protection but also enhance both Th1 and Th2 responses.
  • *P ⁇ 0.05 comparison with the group of 0.5 ⁇ g HA without adjuvant at the same time point.
  • # P ⁇ 0.05 comparison with the group of 5 ⁇ g HA without adjuvant at the same time point.
  • ⁇ 40 means undetectable in an initial dilution of 1:40.
  • Alum-formulated H5N1 influenza vaccines have demonstrated that a prime/boost vaccination schedule is required to generate effective protection against either heterologous or homologous viral strains.
  • the virus-neutralizing antibody levels were below the detection limit for groups that received doses ranging from 0.001 to 3.75 ⁇ g HA antigens with or without alum.
  • the antibody titers increased substantially and virus-neutralizing antibodies were detectable after boosting with the same amount of antigen. It was reported that after the prime/boost immunization with inactivated H5N1, the induced levels of protective antibodies were not statistically different between the groups immunized with 0.2 ⁇ g or 2 ⁇ g HA antigens.
  • PELC-adjuvanted immunization may be particularly helpful in preventing a vaccine shortage since its efficacy:permitted a decrease in antigen dosage and its single-dose formulation eliminated the need for boosting.
  • O/W emulsions can quickly induce a strongly immunocompetent environment at the site of injection, they are more efficient than alum for human vaccination. Furthermore, recent clinical data have demonstrated that pandemic H5N1 vaccines formulated with O/W emulsions induce seroconversion and cross-neutralization superior to that of non-adjuvanted and alum-formulated vaccines.
  • Tween®80 as a hydrophilic emulsifier is that it attacks cell walls and thus is potentially toxic.
  • PEG-b-PLACL One viable alternative is the hydrophilic emulsifier PEG-b-PLACL, which has several advantages over Tween®80.
  • PEG-b-PLACL is derived from the Food and Drug Administration (FDA)-approved PEG, polylactides, and poly(3-caprolactone) and is thus expected to pass all safety tests.
  • FDA Food and Drug Administration
  • polymeric emulsifiers generally result in more stable emulsions and more potent humoral and cellular immune responses than small molecule-emulsified formulations.
  • degradable emulsifiers allow stabilization of emulsion particles during storatte and allow disintegration of the system after the injection.
  • influenza vaccine-induced antibodies decline more rapidly in the elderly. It is believed that the aging population is most susceptible to influenza infection. Recently, an effective vaccination was correlated with the induction of Th1 cytokine IFN- ⁇ , especially in the elderly, who undergo a shift toward Th2 cytokine (such as IL-4) production and a relative reduction in CTL activity as they age. Therefore, increasing IFN- ⁇ induction via vaccination is thought to be an important strategy for overcoming the age-related influenza susceptibility. Toward this end, PELC may be a potent adjuvant due to its stimulation of antigen-specific T-cell proliferation and IFN- ⁇ secretion.
  • PELC-formulated virus also upregulated IFN- ⁇ and IL-4 in splenocytes and increased IgG1 and IgG2a antibody levels more than virus alone.
  • the vaccine formulation with PELC did not significantly skew the immune response toward Th1 or Th2 (Table 6 and FIG. 15B ). These results implied that the antigens adjuvanted with PELC might dramatically enhance the immunogenicity of vaccine candidates. Further investigations are under way to examine the combinations of the PELC vaccine delivery system and immunostimulatory adjuvants, such as CpG oligodeoxynucleotide, in order to manipulate the immune response and alter the Th1/Th2 balance.

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US20160175432A1 (en) * 2014-06-18 2016-06-23 Institute Of Process Engineering, Chinese Academy Of Sciences An oil-in-water emulsion containing no surfactant and use thereof
JP2017522287A (ja) * 2014-06-18 2017-08-10 中国科学院過程工程研究所 界面活性剤を含まない水中油エマルジョン及びその用途
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US10457856B2 (en) 2017-05-12 2019-10-29 Saudi Arabian Oil Company Methods and materials for treating subterranean formations using a three-phase emulsion based fracturing fluid
US10465109B2 (en) 2017-05-12 2019-11-05 Saudi Arabian Oil Company Methods and materials for treating subterranean formations using a three-phase emulsion based fracturing fluid
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US20180327654A1 (en) * 2017-05-12 2018-11-15 Saudi Arabian Oil Company Methods and materials for treating subterranean formations using a three-phase emulsion based fracturing fluid
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JP2020537030A (ja) * 2017-10-04 2020-12-17 ファッション ケミカルズ、 ゲーエムベーハー ウント ツェーオー カーゲーFashion Chemicals, Gmbh & Co Kg ポリ乳酸の新規なエステル及びその組成物
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CN109718372A (zh) * 2017-10-27 2019-05-07 财团法人卫生研究院 用于鼻黏膜的纳米级乳液免疫载剂及其制备方法
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