(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`21 February 2008 (21.02.2008)
`
`
`
`(10) International Publication Number
`
`WO 2008/021123 A1
`
`(51) International Patent Classification:
`B01F 17/42 (2006.01)
`C08G 65/00 (2006.01)
`
`(74)
`
`Agent: OYER, Timothy, J.; Wolf, Greenfield & Sacks,
`P.C., Federal Reserve Plaza, 600 Atlantic Avenue, Boston,
`MA 02210—2206 (US).
`
`(21) International Application Number:
`PCT/USZOO7/0176 1 7
`
`(81)
`
`(22) International Filing Date:
`
`7 August 2007 (07.08.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/836,455
`
`7 August 2006 (07.08.2006)
`
`US
`
`(71) Applicant (for all designated States except US): PRES-
`IDENT AND FELLOWS OF HARVARD COLLEGE
`
`[US/US]; 17 Quincy Street, Cambridge, MA 02138 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): HOLTZE. Chris-
`tian [DE/US]; 16 Bowen Avenue, Medford, MA 02155
`(US). GUERRA, Rodrigo, E.
`[CL/US]; 104 Western
`Avenue, #2, Cambridge, MA 02139 (US). AGRESTI,
`Jeremy [US/US]; 700 Huron Avenue, 14B, Cambridge,
`MA 02138 (US). WEITZ, David, A. [US/US]; 213 Green
`Road, Bolton, MA 01740 (US).
`
`Designated States (unless otherwise indicated, for ever
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, FI, GB, GD, GE, GII, GM, GT, IfN, IIR, IIU, ID, IL,
`1N, 18, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`
`MX, MY, MZ, NA: NG, NI: NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, 117., VC, VN, 7.A,
`ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for ever
`kind of regional protection available): ARIPO (BW, G1 I,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`Published:
`
`with international search report
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`(54) Title: FLUOROCARBON EMULSION STABILIZING SURFACTANTS
`
`
`
`(57) Abstract: Surfactants (e. g., fluorosurfactants) for stabilizing aque
`ous or hydrocarbon droplets in a fluorophilic continuous phase are pre—
`sented. In some embodiments, fluorosurfactants include a fluorophilic
`tail soluble in a fluorophilic (e. g., fluorocarbon) continuous phase, and a
`headgroup soluble in either an aqueous phase or a lipophilic (e.g., hydro—
`carbon) phase. The combination of a fluorophilic tail and a headgroup
`may be chosen so as to create a surfactant with a suitable geometry for
`forming stabilized reverse emulsion droplets having a disperse aqueous
`or lipophilic phase in a continuous, fluorophilic phase. In some embodi—
`ments, the headgroup is preferably nonrionic and can prevent or limit the
`adsorption of molecules at the interface between the surfactant and the
`discontinuous phase. This configuration can allow the droplet to serve,
`for example, as a reaction site for certain chemical and/or biological re—
`actions. In another embodiment, aqueous droplets are stabilized in a flu—
`orocarbon phase at least in part by the electrostatic attraction of two op—
`positely charged or polar components, one of which is at least partially
`soluble in the dispersed phase, the other at least partially soluble in the
`continuous phase. One component may provide colloidal stability of the emulsion, and the other may prevent the adsorption of
`biomolecules at the interface between a component and the discontinuous phase. Advantageously, surfactants and surfactant combi—
`nations of the invention may provide sufficient stabilization against coalescence of droplets, without interfering with processes that
`can be carried out inside the droplets.
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`10
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`FLUOROCARBON EMULSION STABILIZING SURFACTANTS
`
`FIELD OF INVENTION
`
`The present invention relates generally to surfactants, and more specifically, to
`
`surfactants for stabilizing emulsions having a continuous fluor0philic phase.
`
`RELATED APPLICATIONS
`
`This application claims priority to US. Provisional Patent Application No.
`
`60/836,455, filed August 7, 2006, entitled “Fluorocarbon Emulsion Stabilizing
`
`Surfactants,” which is incorporated herein by reference in its entirety.
`
`‘BACKGROUND
`
`Many emulsions comprise an aqueous phase and a hydrocarbon oil phase.
`
`Fluorocarbon oils are often immiscible with both water and hydrocarbon oils; thus, water
`
`or hydrocarbon oils may be dispersed as emulsion droplets in a fluorocarbon phase. The
`
`use of fluorocarbon oils as the continuous phase of an emulsion offers advantages over
`
`conventional hydrocarbon systems. For example, fluorocarbon oils may be well suited
`
`as the continuous phase for emulsions that require reduced diffusion and/or cross-
`
`contamination of hydrophilic or lipophilic material between droplets in the emulsion.
`
`Stabilizing such emulsions, however, sometimes requires the addition of appropriate
`
`surfactants, and these surfactants are often not available or do not have desirable physical
`
`characteristics. This is especially true because fluorocarbons are not commonly used as
`
`the continuous emulsion phase. Accordingly, new surfactants and surfactant systems for
`
`stabilizing droplets of water and hydrocarbon oils or organic solvents in a continuous
`
`fluorophilic phase are needed.
`
`SUMMARY OF THE INVENTION
`
`Surfactants for stabilizing emulsions having a continuous fluorophilic phase are
`
`presented. In one embodiment, a fluorosurfactant is provided. The fluorosurfactant has
`
`one of the general formulas: A—B—A or A-B or (A-B—)n or B—(A)n, or (B)n—A or A—B-A’,
`
`where A (and A’, if present) comprises a fluorophilic component having a molecular
`
`weight greater than 1,000 or greater than 1,500 g/mol, the fluorophilic component
`
`comprising a (per)fluoropolyether or a different poly(perfluoroalkyl—methacrylates), etc.,
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`
`and B is one or both of: a) non—ionic and soluble in an aqueous phase; or b) a
`
`hydrocarbon soluble in a hydrocarbon phase, and n is an integer greater than 0. In other
`
`embodiments, the fluorosurfactant may have a formula such as those described herein.
`
`In another embodiment, a macroemulsion is provided. The macroemulsion
`
`comprises an aqueous dispersed phase or a lipophilic dispersed phase having an average
`
`diameter greater than or equal to about 50 nanometers, a continuous phase comprising a
`
`fluorinated solvent or oil, and a fluorinated, non-ionic surfactant, where at least 95% of
`
`the dispersed phase does not coalesce for at least 30 minutes at 25 degrees C and 1 atm.
`
`In one aspect, the invention is directed to an article. In one set of embodiments,
`
`the article comprises a fluorosurfactant comprising the formula: A-B-A or A—B or (A-B)n
`or B-(A).. or (B)n-A or A-B-A’ or AeX—B or A—Xl-B-Xz-A or (A-Xl-B-X2)n or B-X~(A)n
`or B-(X-A)n or B-(A-X)n or B-Xl-(A-X2)n, wherein A and A’ comprise a fluorophilic
`
`component having a molecular weight greater than 1,000 g/rnol, the fluorophilic
`
`component comprises a fluoropolyether; B is either: a) non—ionic and soluble in an
`
`aqueous phase or b) a hydrocarbon soluble in a hydrocarbon phase; X, when present, is
`
`either a covalent bond or a linking group, and X1 and X2, where present, may be the
`
`same or different; and n is an integer greater than 0.
`
`In another set of embodiments, the fluorosurfactant comprises the formula: A—B—
`A or A-B or (A~B-)n or B-(A).1 or (B)..-A or A-B-A’ or A-X-B or A-Xl-B-XZ-A or (A-
`X‘-B-X2),, or B-X—(A)n or B-(X-A)n or B—(A—X),. or B-Xl-(A-X2)n, wherein A and A’
`
`comprise a component having a molecular weight greater than 1,000 g/mol, the
`
`component comprising a fluorophilic portion having fluorinated side chains; B is either:
`
`a) non-ionic and soluble in an aqueous phase or b) a hydrocarbon soluble in a
`hydrocarbon phase; X, when present, is either a covalent bond or a linking group, and X1"
`
`and X2, where present, may be the same or different; and n is an integer greater than 0.
`
`In still another set of embodiments, the fluorosurfactant comprises the formula:
`
`A—B-A or A-B or (A—B—)n or B-(A).. or (B)n-A or A-B-A’ or A-X-B or A-Xl-B-Xz-A or
`(A—Xl-B—X2)n or B—X—(A)n or B-(X—A)n or B-(A-X)n or B-Xl-(A-X2)n, wherein A and A’
`
`comprise a component having a molecular weight greater than 1,000 g/mol; B is either:
`
`a) non-ionic and soluble in an aqueous phase or b) a hydrocarbon soluble in a
`hydrocarbon phase; X, when present, is either a covalent bond or a linking group, and X1
`
`and X2, where present, may be the same or different; and n is an integer greater than 0.
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`In yet another set of embodiments, the fluorosurfactant has a backbone
`
`comprising the formula: A-B-A or A—B or (A—B-)n or B-(A)n or (B)n-A or A-B—A’ or A—
`X-B or A-X’-B—X2-A or (A-X‘-B—X2)n or 13.—x-(A)n or 13—(X—A)n or B—(A—X)n or B-Xl-
`
`(A-X2)n, wherein A and A’ comprise a fluorophilic component having a molecular
`
`weight greater than 1,000 g/mol, the fluorophilic component comprising a
`
`fluoropolyether; B is either: a) non-ionic and soluble in an aqueous phase or b) a
`
`hydrocarbon soluble in a hydrocarbon phase; X, when present, is either a covalent bond
`
`or a linking group, and XI and X2, where present, may be the same or different; and n is
`
`an integer greater than 0.
`
`In another set of embodiments, the fluorosurfactant having a backbone
`
`comprising the formula: A-B-A or A—B or (A—B—)n or B—(A)n or (B)n—A or A—B—A’ or A-
`X-B or A-X‘-B-X2~A or (A—X1—B—X2 n or B—X-(A)n or B-(X-A)n or B-(A-X)n or B-Xl —
`
`(A-X2)n, wherein A and A” comprise a fluorophilic component comprising a‘
`
`(per)fluoropolyether-, B is either: a) non—ionic and soluble in an aqueous phase or b) a
`
`hydrocarbon soluble in a hydrocarbon phase; X, when present, is either a covalent bond
`
`or a linking group, and X1 and X2, where present, may be the same or different; and n is
`
`an integer greater than 0.
`
`In another aspect, the invention is directed to an emulsion. In one set of
`
`embodiments, the emulsion comprises an aqueous, polar, and/or hydrophilic dispersed
`
`phase or a lipophilic dispersed phase having an average diameter greater than or equal to
`
`about 50 nm; a continuous phase comprising a fluorinated solvent or oil; and a
`
`fluorinated, non-ionic surfactant. In some cases, at least 95% of the dispersed phase does
`
`not coalesce for at least 30 minutes at 25 degrees C and 1 atm.
`
`Inanother set of embodiments, the emulsion comprises an aqueous dispersed
`
`phase or a lipophilic dispersed phase having an average diameter greater than or equal to
`about 50 nm; a continuous phase comprising a fluorinated solvent or oil; and a
`
`fluorinated surfactant comprising a morpholino group. The emulsion, in still another set
`
`of embodiments, includes an aqueous dispersed phase or a lipophilic dispersed phase
`
`having an average diameter greater than or equal to about 50 nm; a continuous phase
`
`comprising a fluorinated solvent or oil; and a fluorinated surfactant comprising a
`
`phosphate group.
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`4
`
`Yet another embodiment is directed to a method. The method, in one some cases,
`
`is generally directed to performing a chemical and/or biological reaction it he dispersed
`
`phase of any of the embodiments described above, or described herein.
`
`In another aspect, the present invention is directed to a method of making one or
`
`more of the embodiments described herein. In another aspect, the present invention is
`
`directed to a method of using one or more of the embodiments described herein.
`
`Other advantages and novel features of the present invention will become
`
`apparent from the following detailed description of various non-limiting embodiments of
`
`the invention when considered in conjunction with the accompanying figures. In cases
`
`where the present specification and a document incorporated by reference include
`
`conflicting and/or inconsistent disclosure, the present Specification shall control. If two
`
`or more documents incorporated by reference include conflicting and/or inconsistent
`
`disclosure with respect to each other, then the document having the later effective date
`
`shall control.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Non-limiting embodiments of the present invention will be described by way of
`
`example with reference to the accompanying figures, which are schematic and are not
`
`intended to be drawn to scale. In the figures, each identical or nearly identical
`
`component illustrated is typically represented by a single numeral. For purposes of
`
`clarity, not every component is labeled in every figure, nor is every component of each
`
`embodiment of the invention shown where illustration is not necessary to allow those of
`
`ordinary skill in the art to understand the invention. In the figures:
`
`FIG. 1A shows an aqueous droplet containing a non-adsorbed biological and/or
`
`chemical species therein, of an aqueous-in—fluorophilic emulsion, according to one
`
`embodiment of the invention;
`
`FIG. 1B shows an aqueous droplet containing an adsorbed biological and/or
`
`chemical species therein, of an aqueous-in-fluorophilic emulsion, according to one
`
`embodiment of the invention;
`
`FIG. 2A shows the combination of a headgroup and a fluorophilic tail to form a
`
`diblock surfactant according to One embodiment of the invention;
`
`FIG. 2B shows the combination of a headgroup and two fluorophilic tails to form
`
`trib10ck surfactant according to one embodiment of the invention;
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`FIG. 2C shows the combination of a headgroup with three fluorophilic tails to
`
`form a multi-block surfactant according to one embodiment of the invention;
`
`FIGS. 2D-2G show various fluorosurfactants including linking moities according
`
`to embodiments of the invention;
`
`FIGS. 3A-3D show various non—limiting illustrative geometries of surfactants
`
`according to one embodiment of the invention;
`
`FIG. 3B shows a non-limiting example of packing geometry of surfactants to
`
`form a droplet of an emulsion according to one embodiment of the invention;
`
`FIG. 4 shows a schematic diagram illustrating adsorption and desorption of
`
`fluorosurfactants during emulsification according to one embodiment of the invention;
`
`FIG. 5 shOWS a schematic diagram illustrating steric stabilization of droplets
`
`against coalescence according to one embodiment of the invention;
`
`FIG. 6 shows a non—limiting example of a device used to form an emulsion
`
`according to one embodiment of the invention;
`
`FIG. 7 shows in-vitro translation inside droplets of an emulsion according to one
`
`embodiment of the invention;
`
`FIGS. 8A and 8B are bright-field and fluorescent micrographs, respectively,
`
`showing fluorescein—labelled polyurethane particles formed by suspension
`
`polymerization according to one embodiment of the invention;
`
`FIG. 9A is a micrograph showing a monodisperse precursor emulsion in a
`
`fluorophilic continuous phase according to one embodiment of the invention;
`
`FIG. 9B is a micrograph showing dried monodisperse polyurethane latex particles
`
`according to one embodiment of the invention;
`.
`FIG. 10A and 10B are bright-field and fluorescent micrographs, respectively,
`
`showing dried, fluorescently labeled, monodisperse particles of polyurethane latex
`
`according to one embodiment of the invention;
`
`FIG. 11A and 1 1B are bright-field and fluorescent micrographs, respectively,
`
`showing cross-linked and fluorescently labeled polyurethane latex particles formed by
`suspension polymerization in a single process step according to one embodiment ofthe
`
`invention;
`
`FIGS. 12A and 12B show cross-linked PU-particles, whose precursor was diluted
`
`with an equal volume of DMSO prior to emulsification according to one embodiment of
`
`the invention;
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`FIGS. 13A and 13B show reinjection and collection of aqueous droplets into a
`
`microfluidic device according to one embodiment of the invention;
`
`FIG. 14 illustrates the formation of droplets by hydrodynamic flow focusing and
`
`the stability of the droplets immediately after emulsification according to one
`
`embodiment of the invention;
`
`FIG. 15 shows monodisperse droplets formed in microfluidic devices containing
`
`viable yeast cells according to one embodiment of the invention;
`
`FIGS. 16A—16C illustrate NMR spectra of certain compounds of the invention;
`
`FIGS. 17A—17H illustrate cells exposed to various surfactants of the invention;
`
`FIG. 18 illustrates the expression of genes in cells exposed to a surfactant, in .
`
`accordance with one embodiment of the invention; and
`
`FIG. 19 illustrates an enzymatic reaction, in accordance with another
`
`embodiment of the invention.
`
`DETAILED DESCRIPTION
`
`Surfactants (e.g., fluorosurfactants) for stabilizing aqueous or hydrocarbon
`
`droplets in a fluorophilic continuous phase are presented (or vice versa). In some
`
`embodiments, the fluorosurfactants include a fluorophilic tail soluble in a fluorophilic
`
`(e. g., fluorocarbon) continuous phase, and a headgroup soluble in either an aqueous
`
`phase or a lipophilic (e.g., hydrocarbon) phase. The headgroup and the tail may be
`
`directly linked, or linked via a linking moiety. The combination of a fluorophilic tail and
`
`a headgroup may be chosen so as to create a surfactant with a suitable geometry for
`
`forming stabilized reverse emulsion droplets having a diSperse aqueous or lipophilic
`
`phase in a continuous, fluor0philic phase. In some embodiments, the headgroup is non-
`
`ionic and can prevent or limit the adsorption of molecules at the interface between the
`
`surfactant and the discontinuous phase. This configuration can allow the droplet to
`
`serve, for example, as a reaction site for certain chemical and/0r biological reactions. In
`
`another embodiment, aqueous droplets are stabilized in a fluorocarbon phase at least in
`
`part by the electrostatic attraction of two oppositely charged or polar components, one of
`
`which is at least partially soluble in the dispersed phase, the other at least partially
`
`soluble in the continuous phase. One component may provide colloidal stability of the
`
`emulsion, and the other may prevent the adsorption of biomolecules at the interface
`
`between a component and the discontinuous phase. Advantageously, surfactants and
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`surfactant combinations of the invention may provide sufficient stabilization against
`
`coalescence of droplets in certain embodiments of the invention, without interfering with
`
`processes that can be carried out inside the droplets.
`
`An “emulsion,” as used herein, is a stable mixture of at least two immiscible
`
`liquids. In general, immiscible liquids tend to separate into two distinct phases. An
`
`emulsion is thus stabilized by the addition of a “surfactant” which functions to reduce
`
`surface tension between the at least two immiscible liquids and/or to stabilize the
`
`interface. In some embodiments, emulsion described herein include a discontinuous or
`
`disperse phase (i.e., the isolated phase stabilized by a surfactant) formed of an aqueous or
`
`lipophilic (e.g., hydrocarbon) substance. The continuous phase may be formed of a
`
`fluorophilic substance (e.g., a fluorocarbon). The present invention involves, in some
`
`embodiments, water—in—fluorocarbon emulsions and hydrocarbon—inefluorocarbon
`
`emulsions having a disperse aqueous or hydrocarbon phase and a fluorocarbon
`
`continuous phase. The isolated disperse aqueous or lipophilic phase in a fluorophilic
`
`solvent can form a “reverse emulsion,” which is simply one example of an emulsion. In
`
`some particular embodiments, the emulsions described herein are macroemulsions.
`
`Macroemulsions are emulsions that are kinetically stable, as compared to
`
`rnicroemulsions, which are thermodynamically stable and undergo spontaneous
`
`formation. In some cases, a microemulsion may include droplets having an average
`diameter of less than about 50 run.
`
`As used herein “droplet” means an isolated aqueous or lipophilic phase within a
`
`continuous phase having any shape, for example cylindrical, spherical, ellipsoidal,_
`
`irregular shapes, etc. Generally, in emulsions of the invention, aqueous and/or lipophilic
`
`droplets are spherical or substantially spherical in a fluorocarbon, continuous phase.
`
`As used herein, “surfactant” defines a molecule that, when combined with a first
`
`component defining a first phase, and a second component defining a second phase, will
`
`facilitate assembly of separate first and second phases. In some cases, a surfactant of the
`
`invention typically can have one or more main fluorophilic chain(s) where one end of the
`
`chain is soluble in the fluorophilic phase of the emulsion and one or more chains that are
`
`not soluble in the fluorophilic phase of the emulsion (e.g., those chains may be soluble in
`
`the aqueous or lipophilic phase). For instance, a surfactant may be a multi-block
`
`surfactant (e. g., ABABABA. . .), where one component of the chain (e.g., “A”) is soluble
`
`in the fluorophilic phase and another component of the chain (e.g., “B”) is soluble in the
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`other phase (e. g., the aqueous or lipophilic phase). As used herein, a multi-block
`
`surfactant is a surfactant having an alternating copolymeric structure or an (A-B—)n
`
`structure, i.e., ABA, ABAB, ABABA, ABABABA, etc.). In some cases, one block may
`
`be soluble in the fluorophilic phase of the emulsion and one block may be soluble in the
`
`other phase of the emulsion (e.g., the aqueous or lipophilic phase). In still other cases,
`
`additional components may be present within the surfactant. For example, a multi-block
`
`surfactant may have other groups present within its polymeric structure, for example,
`
`linking moieties connecting A and B, e.g., (A-X—B-)n, (A-B-X)n, (A-Xl-B-X2)n, or the
`
`like, where “X” represents a covalent bond or a linking moiety, as described below, and
`
`XI and X2, where present, may be the same or different.
`
`As used herein, a “fluorophilic” component comprises any fluorinated compound
`
`such as a linear, branched, cyclic, saturated, or unsaturated fluorinated hydrocarbon. The
`
`fluorophilic. component can optionally include at least one heteroatom (e. g., in the
`
`backbone of the component). In some cases, the fluorophilic compound may be highly
`
`fluorinated, i.e., at least 30%, at least 50%, at least 70%, or at least 90% of the hydrogen
`
`atoms of the component are replaced by fluorine atoms. The fluorophilic component
`
`may comprise a fluorine to hydrogen ratio of, for example, at least 0.2:1, at least 0.5: 1,
`
`at least 1:1, at least 2:1, at least 5:1, or at least 10: 1. In some such embodiments, at least
`
`30%, at least 50%, at least 70%, or at least 90% but less than 100% of the hydrogen
`
`atoms of the component are replaced by fluorine atoms. In other cases, the fluorophilic
`
`component is perfluorinated, i.e., the component contains fluorine atoms but contains no
`
`hydrogen atoms. Fluorophilic components compatible with the present invention may
`
`have low toxicity, low surface tension, and the ability to dissolve and transport gases.
`
`Examples of fluorophilic components are described below.
`
`As mentiOned, in some embodiments, the emulsions of the invention include
`
`discontinuous aqueous and/or 1ipophi1ic(e.g., hydrocarbon) droplets in a continuous,
`
`fluorophilic phase. This means that separate, isolated regions of droplets of an aqueous
`
`and/or lipophilic component are contained within a continuous fluorophilic phase, which
`
`may be defined by a fluorocarbon component. The discontinuous aqueous and/or
`
`lipophilic dr0plets in the nonaqueous phase typically have an average cross-sectional
`
`dimension of greater than 25 nm. In some embodiments, the average cross—sectional
`
`dimension of the droplets is greater than 50 nm, greater than 100 nm, greater than 250
`
`nm, greater than 500 nm, greater than 1 micron, greater than 5 microns, greater than 10
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`microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, or
`
`greater than 500 microns, etc. As used herein, the average cross-sectional dimension of a
`
`droplet is the diameter of a perfect sphere having the same volume as the droplet.
`
`Compositions of the invention are, according to some embodiments, stable for at
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`least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 20
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`minutes, at least about 30 minutes, at least about 40 minutes, at least about 1 hour, at
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`least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, at
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`least about 1 week, at least about 1 month, or at least about 2 months, at a temperature of
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`about 25 degrees Celsius and a pressure of 1 atm. As used herein, a “stable emulsion”
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`means that at least about 95% of the droplets of the emulsion do not coalesce, e.g., to
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`form larger droplets over these periods of time.
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`As used herein, “nonaqueous” is meant to define material such as a fluid that is
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`immiscible with water. That is, a liquid that when mixed with water will form a stable
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`two—phase mixture. The non-aqueous phase need not be liquid, but can be a solid or
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`semi—solid lipid Or other nonpolar substance that is not soluble in water. In some
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`instances, the nonaqueous phase can include a lipophilic component (e.g., a
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`hydrocarbon) or a fluorinated component (e. g., a fluorocarbon). The aqueous phase can
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`be any liquid miscible with water; that is, any liquid that, when admixed with water, can
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`form a room-temperature, single—phase solution that is stable. In some cases, the
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`aqueous phase can comprise one or more physiologically acceptable reagents and/or
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`solvents, etc. Non-limiting examples of aqueous phase materials include (besides water
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`itself) methanol, ethanol, DMF (dimethylformamide), or DMSO (dimethyl sulfoxide).
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`Referring now to FIG. 1A, as a non-limiting illustration, an aqueous-in-
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`fluorophilic (aqueous—in—fluorocarbon) emulsion 5 is shown. The emulsion includes a
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`droplet 10 comprising an aqueous discontinuous phase 20, a fluorophilic (e.g.,
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`fluorocarbon) continuous phase 30, and surfactant molecules 40 at the interface. The
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`surfactant molecules include tail 42 and headgroup 44. Typically, the tail is a
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`fluorophilic chain soluble in the fluorophilic phase of the emulsion and the headgroup is
`soluble in the discontinuous phase. In this particular non-limiting embodiment, the
`headgroup is a hydrophilic component soluble in the aqueous discontinuous phase. The
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`headgroup may be non—ionic in certain embodiments. In other embodiments involving
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`hydrocarbon-in—fluorocarbon emulsions, the headgroup is a lipophilic component soluble
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`in a lipophilic (e.g., hydrocarbon) discontinuous phase.
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`1 0
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`Also shown in the embodiment illustrated in FIG. 1A are components 60 and 62,
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`such as proteins, DNA, and/or cells, which may be contained within the droplet. In some
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`cases, components 60 and 62 are distinguishable. The components may be, for example,
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`reagents, analytes, reactants, etc. to be tested, assayed, and/or reacted within the droplet.
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`In embodiments in which headgroups 44 of the surfactants are non-ionic, adsorption of
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`the components onto the interface between the surfactant and the discontinuous phase
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`may be limited or prevented in some, but not all, cases. Advantageously, this passivation
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`of the interface may allow the components to be investigated as if they were floating in a
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`bulk medium in certain embodiments of the invention, as described in more detail below.
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`In contrast, FIG. 1B shows components 60 and 62 adsorbed onto the interface between
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`the surfactant and the discontinuous phase of the droplet, according to another
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`embodiment of the invention. This adsorption may occur, in some cases, when the
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`headgroup is ionic and/or includes a chemical moiety that preferentially binds and/or
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`adsorbs the components.
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`FIGS. 2A—2C show various non-limiting embodiments of fluorosurfactants of the
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`inVention. As shown in the illustrative embodiment of FIG. 2A, fluorosurfactant 80
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`includes headgroup 82 and fluorophilic component 84. As used herein, a fluorophilic
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`component such as component 84. is referred to as an “A”-block and a non—fluorophilic
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`component of a surfactant, e.g., headgroup 82, is referred to as a “B”-block. The
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`combination of a headgroup with a single fluorophilic component forms an “A—B”
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`structure. The A—B structure is referred to as a diblock structure. In some embodiments
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`of the invention, fluorosurfactants include a multi—block structure, for example, as shown
`
`in FIGS. 2B and 2C. FIG. 2B shows the combination of a headgroup with two
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`fluorophilic components to form a triblock A-B—A structure 86. Structures such as A—B-
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`A’, where A and A’ comprise different fluorophilic components, are also possible.
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`Additional fluorophilic components may be combined with a headgroup to form other
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`multi—block structures, e. g., as shown in FIG. 2C. In some such embodiments,
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`headgroup 82 may be a hydrophilic component that is soluble in an aqueous phase. For
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`example, in some particular embodiments, headgroup 82 may be a non—ionic hydrophilic
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`component, such as a polyether. In other instances, headgroup 82 may be a lipophilic
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`component soluble in a lipophilic (e. g., hydrocarbon) phase. Such an embodiment would
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`be useful for forming hydro carbon-in—fluorocarbon type emulsions. In addition, in some
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`cases, other types of blocks (e.g., having other physical and/or chemical properties) may
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`l 1
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`be included in the multi—block structure, and/or the blocks themselves may each
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`independently have the same or different numbers of repeat units or monomers. For
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`instance, in certain cases, a fluorosurfactant of the invention may comprise random
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`copolymers, terpolymers, and the like.
`
`In another embodiment, a fluorosurfactant of the invention includes a linking
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`moiety (which can be referred to as “X”), which may be hydrophilie or hydrophobic, etc.
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`As shown in FIG. 2D, a moiety 85 may be positioned between the A and B components,
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`e.g., between headgroup 82 and fluorophilic component 84, to produce fluorosurfactant
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`89. In other embodiments, a linking moiety 85 may be positioned between two
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`headgroups, as illustrated in FIG. 2B. FIG. 2F shows a linking moiety 85 attached to two
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`headgroups and a fluorophilic component, and FIG. 2G shows a linking moiety attached
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`to two fluorophilic components and a headgroups. Of course, other configurations are
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`also possible. Linking moieties are described in more detail below.
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`One aspect of the invention involves the formation of stabilized emulsions using
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`fluorosurfactants including those described herein. Surprisingly, in order to obtain long—
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`term stabilized emulsions, certain geometries of the fluorosurfactants are needed in some
`
`cases. For instance, certain ratios of molecular weights of the fluorophilic component to
`
`the headgroup component may be required for steric stabilization of the droplets. In
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`addition, fluorophilic components having large molecular weights can contribute to long
`term colloidal stabilizatiOn, according to certain embodiments. These and other
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`considerations for choosing appropriate components of fluorosurfactants and suitable
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`mixtures of fluorsurfactants may be suitable for forming certain emulsions, for instance,
`emulsions comprising droplets having an average diameter in the micrOn or micrometer
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`range. These and other criteria are described in more detail below.
`
`FIGS. 3A-3E show various non-limiting geometries and packing of
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`fluorosurfactants described herein useful for forming certain emulsiOns having a
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`fluorophilic continuous phase. As illustrated in these embodiments, fluorophilic
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`components having different chemical compositions, molecular weights, and/or lengths
`can contribute to the overall packing geometry of the surfactants (e.g., in the respective
`
`fluorocarbon oil), and, therefore, to the stability of the droplets in the macro emulsion.
`
`The fluorophilic component of surfactant molecules described herein typically
`
`comprise a fluorophilic chain at least C8 in length (i.e., contains at least 8 carbon atoms).
`
`In some embodiments, the fl