Patent Application
20140302516
Fluorescence-based techniques are playing an increasingly important role in the study of biological systems. New fluorescent probes ranging from small organic fluorophores to nanoparticles, such as quantum dots (Qdots), and various forms of genetically encoded green fluorescent proteins (GFPs) have been developed. These fluorescent probes have made new measurements and advances possible but they have their limitations, such as low brightness, insufficient photostability, or toxicity concerns. As a result, there continues to be a need for probes that improve upon the existing fluorescent labels or at least complement them.
Polymer dots (Pdots) have been developed as a new class of fluorescent nanoparticles. Compared to organic dyes and fluorescent proteins, Pdots can possess orders of magnitude greater brightness and are more resistant to photobleaching. When comparing to Qdots, for example, Pdots can be an order of magnitude brighter. Moreover, the dimensions of Pdots can be tuned from several to tens of nanometers without affecting their spectral properties. Pdots with small sizes are desirable in situations where labeling with large nanoparticles may perturb the native behavior of the tagged biomolecules. The small Pdots may also be useful in crowded cellular or intercellular spaces where they can better penetrate and distribute themselves. Various schemes have been developed to control the surface properties and bioconjugation of Pdots, which have provided use of Pdots for cell-surface and subcellular labeling. In addition, Pdot-based ratiometric sensors have been developed, including ones for pH, temperature, and ions, such as iron and copper.
Although Pdots represent a promising new class of fluorescent probes, there is a continued need to for developing methods and compositions involving the use of polymer dots, e.g., methods and compositions for storing polymer dots.
The present invention provides lyophilized polymer dot compositions and related methods.
For example, the present invention includes lyophilized polymer dot compositions including fluorescent nanoparticles, the fluorescent nanoparticles comprising at least one condensed conjugated polymer. The present invention also includes methods of producing polymer dot lyophilized compositions. For example, the methods can include lyophilizing a suspension comprising fluorescent particles, thereby forming the lyophilized composition of fluorescent nanoparticles, wherein the fluorescent nanoparticles are polymer dots each including at least one condensed conjugated polymer.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
The present invention provides lyophilized polymer dot compositions and related methods. The present invention in-part is based on the surprising discovery that polymer dots can be lyophilized and stored while still retaining optical properties, colloidal stability, and, for polymer dot bioconjugates, cell-targeting capability during storage. Polymer dots are fluorescent polymer-based particles and can contain a hydrophobic core and thus potentially may aggregate if water is driven from the core of the polymer dots. While not being limited to any particular theory, during lyophilization, lyoprotectant molecules can form a surface layer, and diffuse into the Pdot and or Pdot shell as water is driven out of the particle. As a result, the colloidal stability and photophysical properties of the particles were retained, and in some instances, improved after lyophilization and being reconstituted into a solution after storage. Lyoprotectant molecules in Pdot and or Pdot shells can, e.g., reduce chain-chain interactions. Therefore, in some instances fluorescence quantum yield was increased and emission bandwidth was narrowed.
The lyophilized compositions and methods can provide several useful results for polymer dots. For example, by using flow cytometry, lyophilized Pdot bioconjugates retained their biological targeting properties and were able to effectively label cells. In one example, cells labeled with lyophilized Pdot bioconjugates composed of PFBT, which were stored for 6 months at −80° C., were ˜22% brighter than those labeled with identical but unlyophilized Pdot bioconjugates. These results among others indicate lyophilization can be a useful approach for storing and shipping Pdot bioconjugates, which is an important practical consideration for ensuring Pdots are widely adopted in biomedical research.
As described further herein, the present invention relates to lyophilized compositions of a new class of fluorescent nanoparticles or polymer dots that have unique properties. By way of background, a brief description of semiconducting polymers is provided to describe some general properties of polymer dots. The majority of organic polymers are insulators. However, when they have π-conjugated structures, electrons can move along the polymer backbone through overlaps in π-electron clouds by hopping, tunneling, and related mechanisms. In general, these π-conjugated polymers can include wide-bandgap semiconductors, the so-called semiconducting polymers.
Organic conjugated polymers and oligomers can be metallic upon heavy doping, a term derived from inorganic semiconductor chemistry. The doping in a conjugated polymer can include an oxidation or a reduction of the π-electronic system and is called p-doping and n-doping, respectively. Semiconducting polymers can exhibit a direct band gap, which leads to an efficient (allowed) absorption or emission at the band edge. Depending on the polymer species, a semiconducting polymer can exhibit strong fluorescence, which can be described in terms of semiconductor band theory. Upon photoexcitation, an electron is excited from the highest occupied energy band (the π band) to the lowest unoccupied energy band (the π* band), thus forming a bound state (exciton) of the excited electron and hole in the π band. The recombination of the excited electron with the hole results in a fluorescent photon. The wavelength of the absorbed light is determined by the π-π* energy gap and can be tuned by altering the molecular structure of the polymer.
Semiconducting polymers have been developed with emission colors that span the full range of the visible spectrum. Important examples of fluorescent semiconducting polymers include polyfluorene (such as PDHF and PFO), poly(phenylene ethynylene) (such as PPE), poly(phenylene vinylene) (such as MEH-PPV and CN-PPV), fluorene-based copolymers (such as PFPV, PFBT, and PF-DBT5), and BODIPY based copolymers, and related derivatives. In many cases, photogenerated electron-hole pairs can dissociate to form free carriers which migrate through the system. The free carriers can either combine to form triplets or deactivate by other nonradiative processes (unwanted processes for fluorescence). They can also be collected to generate electric current (desirable processes for photovoltaics).
As used herein, the term “polymer dot” or “Pdot” refers to a structure including one or more conjugated polymers (e.g., semiconducting polymers) that have been collapsed into a stable sub-micron sized particle. Polymer dots include fluorescent nanoparticles having at least one condensed conjugated polymer. The term “conjugated polymer” is recognized in the art. Electrons, holes, or electronic energy, can be conducted along the conjugated structure. In some embodiments, a large portion of the polymer backbone can be conjugated. In some embodiments, the entire polymer backbone can be conjugated. In some embodiments, the polymer can include conjugated structures in their side chains or termini. In some embodiments, the conjugated polymer can have conducting properties, e.g. the polymer can conduct electricity. In some embodiments, the conjugated polymer can have semiconducting properties, e.g. the polymers can exhibit a direct band gap, leading to an efficient absorption or emission at the band edge. In some aspects, the polymer dots can be described as nanoparticles including at least one condensed (or collapsed) conjugated polymer (e.g., semiconducting polymer) to form the nanoparticle structure. The polymer dots provided herein may be formed by any method known in the art for collapsing polymers, including without limitation, methods relying on precipitation, methods relying on the formation of emulsions (e.g. mini or micro emulsion), and methods relying on condensation. In some embodiments, the polymer dots described herein can be formed by nanoprecipitation.
The polymer dots can be formed using a variety of polymers. Non-limiting examples of semiconducting polymers include fluorene polymers (e.g., Poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF), Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO)), fluorene based copolymers (e.g., Poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV), Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)] (PFBT), Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)] (PFTBT), Poly[(9,9-dioctylfluorenyl-2,7-diyl)-9-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)] (PF-0.1TBT)), phenylene vinylene polymers (e.g., Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV)), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV), BODIPY 570, BODIPY 590, BODIPY 690, and other polymers that are used to make narrow band polymer dots (e.g., BODIPY based polymer dots) such as those described in PCT/US12/71767, which is herein incorporated by reference in its entirety). Other suitable polymers and polymer dots are provided, e.g., in WO2011/057295, which is herein incorporated by reference in its entirety. As provided, e.g., in WO2011/057295, the polymers in the polymer dots can be physically blended or chemically bonded (or chemically crosslinked). For example, the physically blended polymer dots can include polymers that are blended in the polymer dot and held together by non-covalent interactions. Chemically bonded polymer dots can include polymers that are covalently attached to each other in the polymer dot. The chemically bonded polymers can be covalently attached to each other prior to formation of the polymer dots. In some embodiments, the polymers and polymer dots can include those disclosed and claimed, e.g. in PCT/US11/56768. For example, the polymer dots can include those that are directly functionalized and/or have low density functionalization.
In some embodiments, the polymer dots can include a semiconducting copolymer having at least two different chromophoric units. For example, a conjugated copolymer may contain both fluorene and benzothiazole chromophoric units present at a given ratio. Typical chromophoric units used to synthesize semiconducting copolymers include, but are not limited to fluorene unit, phenylene vinylene unit, phenylene unit, phenylene ethynylene unit, benzothiazole unit, thiophene unit, carbazole fluorene unit, boron-dipyrromethene unit, and derivatives thereof. The different chromophoric units may be segregated, as in a block copolymer, or intermingled. As used herein, a chromophoric copolymer is represented by writing the identity of the major chromophoric species. For example, PFBT is a chromophoric polymer containing fluorene and benzothiazole units at a certain ratio. In some cases, a dash is used to indicate the percentage of the minor chromophoric species and then the identity of the minor chromophoric species. For example, PF-0.1 BT is a chromophoric copolymer containing 90% PF and 10% BT.
In certain embodiments, the polymer dots can include a blend of semiconducting polymers. The blends may include any combination of homopolymers, copolymers, and oligomers. Polymer blends used to form polymer dots may be selected in order to tune the properties of the resulting polymer dots, for example, to achieve a desired excitation or emission spectra for the polymer dot.
In some embodiments, the polymer dots can include at least one functional group to facilitate conjugation to other moieties, such as, e.g., biomolecules. As used herein, the term “functional group” refers to any chemical unit that can be attached, such as by any stable physical or chemical association, to the chromophoric polymer, thereby rendering the surface of the chromophoric polymer dot available for conjugation (e.g., bioconjugation). Non-limiting examples of functional groups include, carboxylic acid, amino, mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate, cyanate, succinimidyl ester, alkyne, strained alkyne, azide, diene, alkene, cyclooctyne, and phosphine groups, substituted derivatives thereof, and combinations thereof. In some embodiments, the polymers and/or biomolecules can include a functional group to facilitate conjugations of the polymer dots to the biomolecules.
The lyophilized compositions can include a variety of lyophilization agents (e.g., cryoprotectants and/or lyoprotectants). The constituents can include molecules that are soluble in water, e.g., at a concentration sufficient to provide lyophilization of the polymer dots. As used herein, the term “carbohydrate” refers to, e.g., monosaccharides, oligosaccharides (e.g., disaccharides), and polysaccharides, as well as compounds derived from monosaccharides, oligosaccharides, and polysaccharides.
As used herein, the term “monosaccharide” refers to, e.g., molecules having the general formula: Cx(H2O)y, x≧3. Examples of monosaccharides can include, but are not limited to, glucose, fructose, galactose, xylose, ribose, and the like.
As used herein, the term “oligosaccharide” refers to, e.g., a short monosaccharide polymer that contains, e.g., between 2 to 30 monosaccharide units. An oligosaccharide can include, e.g., a “disaccharide” that refers to, e.g., molecules that are formed when two monosaccharides are joined together and, e.g., a molecule of water is removed. Examples of disaccharides can include, but are not limited to, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, and the like. Other oligosaccharides can include, but are not limited to, trisaccharides (e.g., raffinose), tetrasaccharides (e.g., stachyose), and pentasaccharides (e.g., verbacose).
As used herein, the term “polysaccharide” refers to, e.g., a monosaccharide polymer beyond the length of the oligosaccharide, e.g., a polymer including more than 30 monosaccharide units.
As used herein, the term “sugar alcohol” refers to, e.g., a hydrogenated form of carbohydrate, whose carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Sugar alcohols have the general formula H(HCHO)n+1H. Example sugar alcohols can include, but are not limited to, alditols (e.g., xylitol, mannitol or sorbitol).
The present invention further includes other cryoprotectants and/or lyoprotectants that can be used in the lyophilized compositions provided herein. Example lyoprotectants can include, e.g., glycine, hydroxypropyl-β-cyclodextrin, gelatin and aerosil.
Other constituents can also be included in the solutions and lyophilized compositions described herein. For example, polyethylene glycol or other water soluble polymers can be used. Buffers (e.g., Tris, HEPES, and other known buffers) and salts (e.g., NaCl) can also be used.
As described further herein, the present invention provides lyophilized polymer dot compositions. For example, the present invention includes a lyophilized composition including fluorescent nanoparticles, the fluorescent nanoparticles comprising at least one condensed conjugated polymer. The various conjugated polymers (e.g., semiconducting polymers) are described further herein. In some embodiments, the lyophilized polymer dot compositions can include functionalized polymer dots. The functionalized polymer dots can have at least one functional group available for conjugation (e.g., bioconjugation). In some embodiment, the lyophilized polymer dot compositions can include a polymer dot/biomolecule conjugate, wherein the biomolecule comprises a protein, an antibody, a nucleic acid molecule, a lipid, a peptide, an aptamer, and/or a drug. The biomolecule can be attached to the polymer dot by any stable physical or chemical association. In some embodiments, the lyophilized polymer dot compositions can include a variety of constituents, such as, but not limited to, a monosaccharide, an oligosaccharide (e.g., a disaccharide), and/or a polysaccharide. Sugar alcohols and/or other suitable cryoprotectants and/or lyoprotectants can be used. The constituents added to facilitate lyophilization (e.g., cryoprotectants and/or lyoprotectants), e.g., can be present in a polymer dot solution prior to lyophilization at concentrations ranging between about 1% to about 50%, between about 5% to about 40%, between about 10% to about 30%, between about 1% and about 20%, and between about 10% and about 20%. In some embodiments, several different types (e.g., two or more) of cryoprotectants and/or lyoprotectants can be present at the same or different concentrations in the polymer dots solutions prior to lyophilization.
In one aspect, the present invention includes lyophilized polymer dot compositions including a disaccharide, such as, but not limited to, sucrose, trehalose, maltose, lactose, and any acceptable salt or hydrated forms. In some embodiments, one type of disaccharide is used (e.g., sucrose). In certain embodiments, at least two types of disaccharide can be used (e.g., trehalose and sucrose). The disaccharide(s) can be added to a solution of polymer dots prior to lyophilization. In some embodiments, the concentration of the disaccharide(s) in the solution can vary over a wide range that can be tailored through known techniques to produce useful lyophilized polymer dot compositions that when reconstituted provide polymer dots having about the same particle diameter. In certain embodiments, when the polymer dots in the lyophilized compositions are reconstituted optical properties are the same or improved in comparison to the polymer dots in the solution prior to lyophilization. For example, after lyophilization in combination with a disaccharide, the polymer dots can unexpectedly exhibit the same or increased quantum yield. A variety of concentration ranges can be used for the disaccharides. The disaccharides, e.g., can be present in a polymer dot solution prior to lyophilization at concentrations ranging between about 1% to about 50%, between about 5% to about 40%, between about 10% to about 30%, between about 1% and about 20%, and between about 10% and about 20%. In some embodiments, e.g., sucrose can be present in a polymer dot solution prior to lyophilization at between about 10% w/v to about 20% w/v. Concentrations of the various disaccharides can be optimized using the techniques described herein. For example, a polymer dot solution can be prepared, lyophilized, resuspended, and then the properties (e.g., size) of the polymer dots can be analyzed to confirm that no aggregation occurred due to lyophilization.
The process of lyophilization can be performed in a variety of ways that are generally well known in the art. Lyophilization can, e.g., include a dehydration process used to preserve the polymer dots described herein and to, e.g., make them more convenient for transport. Lyophilization generally works by freezing the polymer dots and then, e.g., reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase. The present invention includes methods for lyophilizing the polymer dots to form lyophilized polymer dot compositions. The lyophilizing can include freezing the polymer dots in aqueous solutions that include a variety of constituents described herein. Freezing can be performed at a variety of temperatures. For example, the polymer dots compositions can be lyophilized by freezing at temperatures at or below about −10° C., at or below about −20° C., at or below about −30° C., at or below about −40° C., at or below about −50° C. at or below about −60° C., at or below about −70° C. or at or below about −80° C.
The concentrations of the polymer dots in the solutions prior to lyophilization can also vary over a wide range. In certain aspects, the concentration of the polymer dots in the solutions prior to lyophilization can depend on the size of the polymer dots. Smaller polymer dots can have a higher concentration than larger polymer dots. In some embodiments, the polymer dots can be present in the solution prior to lyophilization in the millimolar, micromolar, nanomolar, or picomolar range. In some embodiments, the polymer dots can be present between about 1 nM-100 μM, between about 100 nM-1 μM, between about 100 nM-750 nM, between about 100 nM-500 nM, between about 1 nM-500 nM, 1-100 nM, between about 1-75 nM, between about 1-50 nM, between about 1-25 nM, between about 1-20 nM, between about 1-15 nM, between about 1-10 nM, or between about 1-5 nM.
One useful aspect of the lyophilized compositions includes the ability to store the polymer dots for long periods of time. After the storage of the polymer dots over long periods of time as lyophilized polymer dot compositions, the polymer dots can be redispersed in solution and used for a variety of purposes. Advantageously, the polymer dots, e.g., can be redispersed without aggregation, thereby having the same size (e.g., particle diameter) characteristics of the polymer dots prior to lyophilization. Suitable storage periods can include, but are not limited to, longer than one day, longer than one week, longer than one month, longer than two months, longer than three months, longer than six months, or longer than one year. In some embodiments, the storage period can range from about one day to about one year, from about one day to about six months, from about one day to about three months, from about one day to about two months, or from about one day to about one month.
Another useful aspect of the lyophilized compositions includes the ability to improve the photophysical properties of the Pdots through a lyophilization process. Pdots can contain hydrophobic core and thus the chain conformation may change if water is driven from the system. In case of using lyoprotectants during lyophilization, lyoprotectant molecules can form a surface layer, and diffuse into the Pdot shell as water is driven out, therefore reducing polymer chain-chain interactions. As a result, the photophysical properties of the Pdots can be improved after lyophilization and being reconstituted into a solution after storage. In some embodiments, fluorescence quantum yield can be increased. In some embodiments, the fluorescence emission bandwidth can be reduced. In some embodiments, the cell labeling brightness can be increased.
In one aspect, the present invention further includes methods for preparing lyophilized polymer dot compositions. The methods can include lyophilizing the polymer dots in a variety of solutions described herein. Lyophilizing the polymer dots in solution can include freezing the polymer dot solutions at any suitable temperature to produce a lyophilized polymer dot compositions. For example, the polymer dots compositions can be lyophilized by freezing at temperatures at or below about −10° C., at or below about −20° C., at or below about −30° C., at or below about −40° C., at or below about −50° C. at or below about −60° C., at or below about −70° C. or at or below about −80° C. In some aspects, the methods, e.g., can include providing a solution including polymer dots in combination with other constituents, and freezing the solution at a desired temperature, e.g., at about −80° C. or −20° C. for a period of time. The lyophilized polymer dots can be redispersed by combining the lyophilized polymer dots with a solution.
In some embodiments, the present invention includes method of producing a lyophilized composition including lyophilizing a suspension of polymer dots, thereby forming the lyophilized composition of polymer dots, wherein the polymer dots are fluorescent nanoparticles including at least one condensed conjugated polymer. In some embodiments, the methods can include, e.g., (a) combining (i) a liquid including polymer dots with (ii) a first aqueous solution, thereby forming a first suspension comprising the polymer dots; and (b) lyophilizing the suspension, thereby forming the lyophilized composition of polymer dots, wherein the polymer dots are fluorescent nanoparticles including at least one condensed conjugated polymer.
In some embodiments, the present invention includes method of producing a lyophilized composition including polymer dot with reactive functional groups. The reactive functional groups can include amine reactive functional group such as succinimidyl ester, sulfhydryl reactive functional group such as maleimide, or reactive functional group for click chemistry such as alkyne, azide, strained alkyne, cyclooctyne, and phosphine groups. In some embodiments, the methods can include, e.g., (a) combining (i) a solution of conjugated polymer with reactive functional groups in good solvent with (ii) a poor solvent, followed by evaporation of the poor solvent, thereby forming a first suspension comprising the polymer dots; and (b) lyophilizing the suspension, thereby forming the lyophilized composition of polymer dots with reactive functional groups, wherein the polymer dots are fluorescent nanoparticles including at least one condensed conjugated polymer. Good solvents can include, e.g., a solvent in which the conjugated polymer is soluble without forming a polymer dot. Poor solvents can include, e.g., a solvent in which the conjugated polymer is poorly soluble and thereby forms polymer dots after introduction into the poor solvent.
The lyophilization methods above can be used, e.g., to produce amine-reactive Pdots such as Pdots with succinimidyl ester. These Pdots, e.g., can be directly mixed with a biomolecule (e.g., a protein) to form polymer dot bioconjugates. In some embodiments, EDC/NHS can be used to activate a Pdot-COOH to form Pdot-NHS, after which lyophilization can be performed. In certain embodiments, NHS-terminated conjugated polymer can be synthesized, injected into a solution of methanol or ethanol to form Pdots. The Pdots can then be lyophilized to produce lyophilized Pdot-NHS.
In another aspect, the present invention provides kits including the lyophilized polymer dot compositions. A typical kit of the invention includes a unit dosage form of a lyophilized polymer dot composition of the present invention, e.g., in a sealed container. In one embodiment, the kit further comprises a sealed container of a suitable vehicle in which the polymer dot composition can be dissolved to form a particulate-free sterile solution that is suitable for administration or use.
This example describes an example lyophilization, a freeze-drying/dehydration technique, that can be used to prepare Pdot bioconjugates for long-term storage or shipping, provided the right conditions are used. Lyophilization is an important practical advance for making Pdots practical to use in biomedical research.
Materials. Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-(2,10,3)-thiadiazole)] (PFBT; MW, 157 000 Da; polydispersity, 3.0), Poly(9,9-dioctylfluorenyl-2,7-diyl) end capped with dimethyl phenyl (PFO, MW 120000 Da), poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-(2,1′,3)-thiadiazole)]10% benzothiadiazole (PF10BT, MW 100000 Da) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CNPPV, MW 15000 Da) were purchased from American Dye Source Inc (Quebec, Canada). PFBT directly functionalized with carboxylic acid (PFBT-COOH) groups was synthesized in our lab. Polystyrene-grafted ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH; MW 21,700 Da of PS moiety; 1200 Da of PEG-COOH; polydispersity, 1.25) were purchased from Polymer Source Inc. (Quebec, Canada). Sucrose was ordered from Avantor Performance Materials (Phillipsburg, N.J., USA). Streptavidin was purchased from Invitrogen (Eugene, Oreg., USA). Bovine serum albumin (BSA) and ethylcarbodiimide hydrochloride (EDC) were bought from Sigma (St. Louis, Mo., USA).
Streptavidin conjugation of Pdots. Pdots were prepared using a nano-reprecipitation method as reported earlier. Wu et al., J. American Chem. Soc. 132: 15410-15417 (2010). Briefly, a tetrahydrofuran (THF) solution containing 50 μg/mL of semiconducting polymer (PFBT, CNPPV, PFO or PF10BT) and 16 μg/mL of PS-PEG-COOH was prepared. A 5-mL aliquot of the mixture was quickly injected into 10 mL of water under vigorous sonication. THF was removed by blowing nitrogen gas into the solution at 90° C. The THF-free Pdot solution was sonicated for 1-2 minutes and filtrated through a 0.2-μm cellulose membrane filter. For PFBT-COOH Pdots, no additional PS-PEG-COOH was added. THF solution containing only 50 μg/mL PFBT-COOH was injected into water directly. In a typical conjugation reaction, 80 μL of polyethylene glycol (5% w/v PEG, MW 3350) and 80 μL of HEPES buffer (1M, PH 7.3) was added to 4 mL of Pdot solution. Streptavidin (1 mg/mL, 30 μL) was then added to the solution and mixed well. Finally, 80 μL of freshly-prepared EDC solution (5 mg/mL in MilliQ water) was added to the solution, and the mixture was magnetically stirred for 4 hr at room temperature. The resulting Pdot conjugates were finally concentrated in a spin column (100K MW) and were purified with a Bio-Rad Econo-Pac 10DG column (Hercules, Calif., USA). After purification, the proper amount of BSA was added to reach a final concentration of 1% (w/v). The hydrodynamic sizes of Pdots were measured with a dynamic light scattering (DLS) spectrometer (Malvern Zetasizer Nano ZS, Worcestershire, United Kingdom). Fluorescence quantum yields were collected using an integrating sphere (model C9920-02, Hamamatsu Photonics) with proper wavelength excitation. Fluorescence spectra of Pdots were taken with a Fluorolog-3 fluorospectrometer (HORIBA JobinYvon, NJ, USA).
Lyophilization. Solutions of streptavidin-conjugated Pdots or unconjugated Pdots were prepared at two different concentrations (4 nM and 20 nM) by diluting the Pdot solution with a buffer that was composed of 20 mM HEPES (pH 7.3), 0.1% (w/v) PEG and 0.05% (w/v) BSA. For PFBT, Pdots at 100 nM concentration were also prepared. Sucrose was added to reach the desired final concentrations (10%, w/v). The stock Pdot solutions were aliquoted into several vials. Half of them were rapidly frozen in liquid nitrogen for 2 minutes, and were immediately placed under vacuum on a Labconco Freezone 6 freeze-dryer (Kansas City, Mo., USA). After ˜18 hr, lyophilized samples were removed from the freeze-dryer and were labeled as “lyophilized Pdots” and stored at −80° C. for a desired amount of time (from one day to 6 months). The other half of the aliquots were labeled “unlyophilized” and were placed in a 4° C. refrigerator.
Cell culture. The breast cancer cell line, MCF-7, was ordered from American Type Culture Collection (ATCC, Manassas, Va., USA). Cells were cultured at 37° C., 5% CO2 in Eagles minimum essential medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% Pen Strep (5000 units/mL penicillin G, 50 μg/mL streptomycin sulfate in 0.85% NaCl). Cells were cultured prior to experiments until confluence was reached. The cells were harvested from the culture flask by briefly rinsing with culture media followed by incubation with proper amount of Trypsin-EDTA solution (0.25% w/v Trypsin, 0.53 mM EDTA) at 37° C. for 5 minutes. After complete detachment, cells were rinsed, centrifuged, and re-suspended in the culture media. Their concentration was determined by microscopy using a hemacytometer.
Immunofluorescence. For labeling cell-surface markers with IgG conjugates, a million MCF-7 cells in 100 μL labeling buffer (1×PBS, 2 mM EDTA, 1% BSA) were incubated with 0.3 μL of 0.5 mg/mL biotinylated primary anti-human CD326 EpCAM antibody (eBioScience, San Diego, Calif., USA) on a rotary shaker in the dark and at room temperature for 30 minutes. This was followed by a washing step using the labeling buffer. The cells were incubated with 4 nM streptavidin-conjugated Pdots (diluted from the 20 nM Pdots solution) in BlockAid™ blocking buffer (Invitrogen, Eugene, Oreg., USA) for 30 minutes on a shaker in the dark and at room temperature, followed by two washing steps with the labeling buffer. Negative controls were obtained by incubating cells with streptavidin-conjugated Pdots without any previous incubation with the primary biotinylated antibody. Cell fixation was performed afterwards by dissolving the cell pellet obtained by centrifugation in 500 μL of fixing buffer (1×PBS, 2 mM EDTA, 1% BSA, 1% paraformaldehyde).
Flow cytometry experiments. Measurements were performed on labeled cell samples containing 106 cells/0.5 ml and prepared as previously described. Wu et al., Angewandte Chemie-Intl. Ed., 49:9436-9440 (2010). The flow cytometer BD FACSCanto II (BD Bioscience, San Jose, Calif. USA) was used. Cells flowing in the detection chamber were excited by a 488-nm laser light. Side- and forward-scattered light were collected and filtered by a 488/10 nm band-pass filter, while fluorescence emission was collected and filtered by a 502-nm long-pass and a 530/30 nm (for PFBT and PFBT-COOH) or a 582/42 nm (for CNPPV) band-pass filter. All signals were detected by photomultiplier tubes. For all flow experiments, representative populations of detected cells were chosen by selecting an appropriate gate. Detection of cell fluorescence was continued until at least 104 events had been collected in the active gate.
Size. We chose streptavidin conjugated PFBT Pdots (Strep-PFBT) to optimize the lyophilization recipe. The particular Strep-PFBT Pdots we prepared had a hydrodynamic diameter of 32 nm (
We modified our lyophilization recipe. First, we added 1% (w/v) sucrose. Here, we found the hydrodynamic diameter of the rehydrated Pdots was 50 nm (
Table 1. The hydrodynamic diameter of lyophilized and unlyophilized streptavidin conjugated and unconjugated Pdots after long term storage. Strep-PFBT-COOH Pdots were stored at −80° C. for 1 month. All the other Pdots were stored for 6 months. All lyophilized Pdots were rehydrated with water. Both lyophilized and unlyophilized Pdots were measured with dynamic light scattering without sonication. (Lyoph.: lyophilized; Unlyoph.: unlyophilized.)
Optical property. The optical properties of both lyophilized and unlyophilized Pdots were measured and compared. We focused on the absorption spectra, emission spectra, and quantum yield. We measured the absorption and emission spectra of lyophilized Pdots after 6 months storage and compared them with that of their unlyophilized counterparts. As shown in
We next studied the brightness of Pdots to determine if lyophilization had a negative effect on their fluorescence intensity. To facilitate the brightness comparison between lyophilized and unlyophilized Pdots of different concentrations, we measured their quantum yield (QY), because quantum yield is less dependent on concentration than fluorescence intensity as shown below:
F=α*I*Q*n (1)
Where F is fluorescence emission intensity; α is the instrument factor; I is the excitation intensity; Q is the quantum yield; n is the concentration of Pdots. Table 2 shows the quantum yield values of various Pdots that had and had not undergone lyophilization. First, we found that the conjugated streptavidin molecules did not affect the quantum yield of Pdots. The quantum yield values of streptavidin conjugated CNPPV and PFBT Pdots stayed at similar levels as their corresponding unconjugated Pdots. For example, the QY values of 4 nM lyophilized Strep-PFBT and unconjugated PFBT Pdots after 6 months storage were both 33%. Second, the quantum yield of lyophilized Pdots did not fluctuate much among different concentrations of Pdots. For example, the QY values of 4 nM lyophilized and 20 nM lyophilized PFO Pdots after 6 months storage were both 47%.
More importantly, the quantum yield of most unlyophilized Pdots decreased after long term storage, but the quantum yield of lyophilized Pdots remained at the same level for the duration of the storage time. For example, the quantum yield of 20 nM lyophilized Strep-PFBT Pdots was 37% after stored for 1 day and 36% after 6 months storage, respectively. In contrast, the quantum yield of most unlyophilized Pdots showed a small but consistent decrease: when the same Pdots (20 nM Strep-PFBT Pdots) were stored unlyophilized, the quantum yield decreased from 33% to 30% after 6 months storage. Similar quantum yield changes were also found in unconjugated PFBT and PFO Pdots. It is likely that oxidation of the semiconducting polymer, which would reduce the quantum yield, was minimized when the Pdots were lyophilized and stored at −80° C.
These results demonstrate that the brightness of Pdots certainly was not adversely affected by the lyophilization process, and remarkably, there could even be an enhancement in the optical performance of Pdots by going through the lyophilization procedure. For example, the quantum yield enhancement of 4 nM lyophilized Strep-PFBT Pdots over unlyophilized Strep-PFBT Pdots after 1 day storage was (0.35−0.32)/0.32=9.4%. Although the mechanism that underlies this increase in quantum yield caused by lyophilization is unclear, we think the lyophilization process caused the internal rearrangement of the backbone or internal packing of the semiconducting polymer.
Table 2. The quantum yield values of lyophilized and unlyophilized Pdots stored for up to 6 months. For each Pdot, samples with two concentrations (4 nM and 20 nM) were tested. (1 D: 1 day; 1 M: 1 month; 6 M: 6 months.)
Labeling efficiency. Once we found the conditions where Pdots retained their colloidal stability after lyophilization and confirmed that the brightness of lyophilized Pdots did not decrease, we tested the cell targeting capability of the Pdot bioconjugates to ensure they maintained their biological specificity. We used streptavidin-conjugated Pdots to label the cell surface receptor, EpCAM, which is an epithelial cell adhesion marker currently used for the detection of circulating tumor cells. We used flow cytometry to quantify the brightness of the cell labeling and the degree of non-specific absorption. For comparison between lyophilized and unlyophilized Pdot-streptavidin, we used 20 nM streptavidin conjugated Pdots (Strep-CNPPV, Strep-PFBT and Strep-PFBT-COOH) with 10% sucrose. The labeling efficiency of both lyophilized and unlyophilized Pdot-streptavidin conjugates stored for up to 6 months was tested.
We then compared the non-specific absorption and positive cell labeling by Pdot-streptadvidin that had been lyophilized and stored over various time versus Pdots that were not lyophilized. Samples stored for 1 day after lyophilization were first tested. This experiment reports on any potential effect caused by undergoing the lyophilization process. As shown in the top panels in
The data also show that the intensity peaks for cells labeled with both lyophilized (red curve) and unlyophilized (green curve) Pdots in the presence of primary antibody (positive) were well separated from that of the negative control samples. Specifically, for Strep-CNPPV and Strep-PFBT-COOH Pdots, the positive peak intensity values of lyophilized Pdots were similar to that of unlyophilized Pdots. For Strep-PFBT Pdots, the peak intensity value of lyophilized Pdots was a little larger than that of unlyophilized Pdots. The labeling brightness difference is consistent with our measured quantum yield values: for 20 nM Strep-CNPPV and Strep-PFBT-COOH, which were stored for 1 day, the QY values of lyophilized Pdots were similar to that of unlyophilized Pdots (table 2); for Strep-PFBT, the cell labeling enhancement is 10%, which is similar to our measured quantum yield enhancement (9%). This result indicates that the lyophilization process did not impair the performance of Pdot bioconjugates, and for some semiconducting polymers, even enhanced their performance.
We further tested the cell labeling efficiency of lyophilized and unlyophilized Pdots after long term storage. As displayed in the bottom panels in
Control Experiment. Here, we used flow cytometry to quantify the brightness of Pdot-tagged MCF-7 cells, where the cells were labeled using Pdot solutions that contained either 10% or 0% sucrose.
The flow data was collected with a commercial BD FACSCanto II cytometer (BD Bioscience, San Jose, Calif. USA). Cells were illuminated by 488-nm laser light and fluorescence emission was filtered by a 502-nm long pass filter before being detected by an array of photomultiplier tubes (PMTs).
We studied the effect of lyophilization on the properties of Pdot-streptavidin bioconjugates, including colloidal stability, spectral properties, brightness, and labeling efficiency. Samples of various concentrations were stored for up to 6 months. We found that lyophilization with 10% sucrose was a good strategy to preserve Pdot bioconjugates. The rehydrated Pdots after lyophilization had the same size as that before lyophilization, even in the absence of sonication to help re-disperse the Pdots. The lyophilization procedure did not negatively affect the optical properties of Pdots. The quantum yield values of lyophilized Pdots using sucrose showed a consistent, albeit small, improvement in quantum yield after lyophilization; this phenomenon is likely caused by the rearrangement of the polymer backbone or internal packing during lyophilization. The use of other lyoprotectant molecules other than sucrose can result in much more significant increase in quantum yield (see Example 2), which is an important finding because Pdots with high quantum is highly desired for improving the brightness of the probe for a wide range of application. In addition to improving quantum yield, the use of appropriate lyoprotectant molecules can also result in a narrowing of the emission spectrum of the Pdot (
The brightness of cells labeled with lyophilized Strep-PFBT Pdots stored for 6 months showed a 22% enhancement over the unlyophilized counterpart, likely because oxidation of the semiconducting polymer was minimized when the Pdots were lyophilized and stored at −80° C. We believe lyophilization will be a preferred route for the long-term storage of Pdots, which makes it an important practical consideration for the wide-spread adoption of bioconjugated Pdots in biomedical research.
This example describes the effect of sucrose concentration on the lyophilization of Pdots. We used a series of sucrose concentrations (0%, 1%, 10%, 20%, 50%) (w/v) to lyophilize Pdot. Two types of Pdots were used. One is PFBT (Mw=73 k)+30% (w/w) PS-PEG-COOH, and the other is the directly functionalized PFBT-COOH 2%. The Pdots were prepared using nanoprecipitation as described in Example 1. The samples had a size of 21 nm at 20 nM concentration in aqueous solution. Different concentration of sucrose was added to the Pdot aqueous solution. The sample was then lyophilized. After lyophilization, the sample was stored in −80° C. freezer for 1 day and then re-dispersed in aqueous solution.
Size and quantum yield were measured to describe whether there is any change of the Pdot after lyophilization (
This example describes the use of several lyophilization agents in the application of Pdot lyophilization. The direct functionalized PFBT-COOH 2% Pdots and BODIPY-690 were prepared using nanoprecipitation method as described in Example 1. Both the as-prepared Pdots had a size of 20 nm and at a concentration of 20 nM in aqueous solution. Different lyophilization agents with 5-20% (w/v) were added to the Pdot aqueous solution. In the combination of lyophilization agents, the total agents concentration was 10% (w/v) in the solution. The sample with lyophilization agent was then lyophilized. After lyophilization, the sample was stored in −80° C. freezer for 1 day and then re-dispersed to aqueous solution.
Size and quantum yield were measured to describe whether there was any change of the Pdots after lyophilization. The results were shown in the lower panel in
We also lyophilized the Pdots with BSA, and the result showed that the quantum yield of the lyophilized Pdots was also generally higher than that of unlyophilized Pdots. The size of the lyophilized Pdots by DLS was not recorded due at least in-part to the presence of large amounts of BSA in the solution, which can interfere with DLS measurements. There was a strong peak at around 3 nm that corresponded to the size of BSA molecules. But both lyophilized and unlyophilized Pdots solution were very clear, indicating no obvious aggregation was formed during the lyophilization process with BSA.
This example describes the lyophilization of three types of BODIPY based Pdots with centered emission at 570 nm, 590 nm and 690 nm. It shows the lyophilized BODIPY Pdots can be stored for at least 6 months.
The optical properties of both lyophilized and unlyophilized Pdots were measured and compared. We measured the absorption and emission spectra of lyophilized Pdots after 6 months storage and compared them with that of their unlyophilized counterparts. As shown in
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 61/785,293, filed Mar. 14, 2013, which application is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61785293 | Mar 2013 | US |
