WO2016179260A1 - Ultrasmall nanoparticles and methods of making and using same - Google Patents
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- WO2016179260A1 WO2016179260A1 PCT/US2016/030752 US2016030752W WO2016179260A1 WO 2016179260 A1 WO2016179260 A1 WO 2016179260A1 US 2016030752 W US2016030752 W US 2016030752W WO 2016179260 A1 WO2016179260 A1 WO 2016179260A1
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Classifications
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
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- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
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- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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- A61P35/00—Antineoplastic agents
Definitions
- the disclosure generally relates to ultrasmall nanoparticles and methods of making and using same. More particularly the disclosure generally relates to ultrasmall silica and aluminosilicate nanoparticles.
- SNPs Silica nanoparticles
- Particles >12nm are not effectively cleared from the body in vivo and unfavorably distribute to the liver and other organs/tissues, potentially exposing these tissues to toxic elements (especially if these >10nm SNPs are modified with drugs and/or radioactivity). Particles about 8nm in diameter reside in the body for about a day, 10-1 lnm about 3-5 days, but if greater than 12nm do not clear or clear very slowly.
- SNPs polyethylene glycol coated (PEGylated) fluorescent core-shell silica nanoparticles
- FDA U.S. Food and Drug Administration
- IND investigational new drug
- compositions are of interest leading to higher rigidity of the organic dye environments as increases in rigidity have directly been correlated with increases in per dye fluorescence yield as a result of decreases in non-radiative rates.
- silica compositions derived from aluminum alkoxides as additives are particularly interesting as they are known hardening components in alkoxysilane derived silica and alumina is an approved adjuvant added to high-volume vaccinations injected intramuscularly and subcutaneously.
- fluorophores including near infrared (NIR) emitters
- NIR near infrared
- Brightness can be enhanced via addition of extra silica shells.
- This methodology further enables synthesis of ⁇ 10 nm sized fluorescent core and shell SNPs with previously unknown compositions.
- an aluminum sol-gel precursor leads to fluorescent aluminosilicate nanoparticles (ASNPs) and core-shell ASNPs.
- ASNPs fluorescent aluminosilicate nanoparticles
- Encapsulation efficiency and brightness of highly negatively charged NIR fluorophores is enhanced relative to the corresponding SNPs without aluminum.
- Resulting particles show quantum yields of -0.8, i.e. starting to approach the theoretical brightness limit.
- All particles may be PEGylated providing steric stability.
- PEGs can be employed to introduce ligands onto the PEGylated particle surface of fluorescent SNPs, core-shell SNPs, and their aluminum containing analogues, producing ligand functionalized ⁇ 10 nm NIR fluorescent nanoprobes.
- fluorescent SNPs fluorescent SNPs
- core-shell SNPs and their aluminum containing analogues
- ligand functionalized ⁇ 10 nm NIR fluorescent nanoprobes producing ligand functionalized ⁇ 10 nm NIR fluorescent nanoprobes.
- the SNPs and ASNPs described here and synthesized in water will be referred to as Cornell prime dots or C dots and A1C dots.
- C dots and AIC dots can range from about 2.4nm to about 7.3nm or greater.
- Conditions which may vary include: the temperature of the reaction; the concentration of TMOS or comparable compound; the concentration of base (e.g., ammonium hydroxide) and/or the particle growth period. Examples of these conditions are included in Table 1 of Example 1.
- the temperature can range from about RT to about 80°C or greater.
- the concentration of TMOS can range from about 0.011M to about 0.043M or greater.
- the concentration of ammonia hydroxide can range from about 0.002M to about 0.06M or greater.
- the particle growth period can range from about 10 minutes to about 20 hours or greater.
- Fig. 1 is an illustration of water based fluorescent SNP growth pathways together with the chemical structures of produced particles.
- Fig. 2 shows characterization of blank SNPs (i.e. without fluorophore encapsulation)
- a-j show TEM images at two different magnifications (insets) of blank SNPs with the following average diameters as measured by DLS in aqueous solution: (a) 2.4 nm, (b) 3.1 nm, (c) 3.7 nm, (d) 4.2 nm, (e) 4.5 nm, (f) 5.2 nm, (g) 5.9 nm, (h) 6.5 nm and (i) 7.3 nm.
- (k) shows FCS autocorrelation curves; (1) shows absorbance (matched) and emission spectra; (m) shows FCS derived number of fluorophores per particle; (n) shows FCS measured (columns) and calculated (dots) fluorescence brightness per particle of fluorescent SNPs with varying average diameters as compared to free dye in aqueous solution.
- Fig. 3 shows TEM images at two different magnifications (insets) of Cy5- encapsulated fluorescent core-shell SNPs (C dots) with 0 (core/seed) to 4 shells, and the following average diameters as measured by FCS: 5.0 nm (core/seed), 6.1 nm, 6.8 nm 7.8 nm and 8.7 nm (a-e).
- Figure 4 shows absorbance (matched) and emission spectra (left) as well as
- FCS characterization (right) of C dots derived from different types of fluorophores (a-f).
- RhG RhG
- TMR TMR
- Cy5 Cy5.5
- e DY782
- CW800 Normalized absorbance and emission spectra of C dots with different dyes/colors
- h A photo showing the solution appearance of C dots derived from different color dyes. From left to right: RhG, TMR, Cy5, Cy5.5, DY782 and CW800.
- Figure 5 shows (a) Solid-state 29 Si CP/MAS (top) and 27 Al MAS (bottom) NMR spectra of A1C dots with and without silica shells; the insert on the right shows a model of the molecular structure of the corresponding aluminosilicate network together with covalently encapsulated NIR Cy5.5 dye. (b) TEM image of core ASNPs. (c) TEM image of core-shell ASNPs.
- Figure 6 shows (a) Conjugation chemistry of c(RGDyC) functionalized PEG- silane. (b) Absorbance spectra comparing free c(RGDyC) peptide, free Cy5 dye and Cy5-C dots (no silica shell) with/without c(RGDyC) surface functionalization suggesting successful c(RGDyC) surface modification, (c) Absorbance spectra comparing free c(RGDyC) peptide and different types of c(RGDyC) surface functionalized C dots suggesting success of nanoparticle-PEG-surface functionalization with ligands is independent of particle architecture (i.e. with or without extra silica shells), types of fluorophores encapsulated, or particle composition (i.e. C dots versus A1C dots).
- Figure 7 shows a molecular-graphics rendering of Cy5 fluorophore, PEG- silane, maleimide-functionalized PEG-silane, c(RGDyC) peptide, SiCte matrix (a) and a c(RGDyC) -PEG-Cy5-C dot (b and c).
- the C dot model consists of a ⁇ 3nm silica core which encapsulates one Cy5 fluoro-phore, a ⁇ 1 nm silica shell, a -1.5 nm PEG layer and 16 easily accessible c(RGDyC) ligands.
- Fig. 8 shows in-situ FCS measurement results, (a) particle diameter and (b) particle concentration during the reaction and particle growth.
- Fig. 9 shows TEM images of (a) particles synthesized at room temperature
- Fig. 10 shows 6.2 nm C dot FCS characterization result as an example of a fit of a FCS autocorrelation curve.
- Fig. 11 shows TEM images of Cy5-encapsulated fluorescent and PEGylated
- Fig. 12 shows TEM images of blank PEGylated core-shell SNPs (without fluorophores) with 0-3 layers of shells (a-d).
- the average diameters are measured by DLS.
- e Diameter distribution of SNPs with 0-3 layers of shells as measured by DLS.
- Fig. 13 shows switching the particle growth mechanism in a single synthesis.
- the growth of the forming particles first goes through controlled aggregation followed by monomer addition.
- Three sets of particles are produced from this synthesis through quenching the particle growth at different reaction time points via addition of PEG-silane.
- the resulting particles are characterized by (b) FCS and (c) optical absorbance / emission measurements.
- Fig. 14 shows DLS size distribution and TEM of ASNPs with average diameter around 3.5 nm.
- Fig. 15 shows a GPC elugram of as-synthesized Cy5 doped PEGylated SNPs
- Fig. 16 shows a comparison of FCS measured autocorrelation curves of free
- Cy5 dye Cy5-C'dots (core only), and c(RGDyC) functionalized Cy5-C dots (c(RGDyC)- Cy5-C dots).
- Fig. 17 shows a TGA curve for a 6 nm (measured by DLS) blank non- fluorescent PEGylated SNP (without fluorophore encapsulation).
- the present disclosure provides nanoparticles (e.g., core or core-shell nanoparticles).
- the nanoparticles are also referred to herein as ultrasmall nanoparticles.
- the present disclosure also provides methods of making and using the nanoparticles.
- the techniques disclosed herein provide an aqueous synthesis approach to ultrasmall functional PEGylated fluorescent silica nanoparticles with improved control in multiple aspects, including particle size, particle size distribution, fluorescence wavelength, fluorescence brightness, compositions, particle PEGylation, particle surface functionalization, synthesis yield, product purity and manufacture reliability.
- the systematic and precise control covering all these aspects in a single organic-inorganic hybrid nanomaterials synthesis system has never been achieved before, preventing the safe translation of organic-inorganic hybrid nanomaterials from the laboratory to the clinic. Therefore, the techniques disclosed herein provide access to well-defined and systematically highly tunable silica-based nanomaterials that show significant potential in nanomedicine applications.
- the present disclosure provides a method of making ultrasmall nanoparticles.
- the methods are based on use of aqueous reaction medium (e.g. water).
- the nanoparticles can be surface functionalized with polyethylene glycol groups (e.g.,
- the methods as described herein can be linearly scaled up, e.g., from 10 ml reaction to 1000ml or greater without any substantial change in product quality. This scalability is important for large scale manufacture of the nanoparticles.
- the methods are carried out in an aqueous reaction medium (e.g., water).
- the aqueous medium comprises water.
- Certain reactants are added to the various reaction mixtures as solutions in a polar aprotic solvent (e.g., DMSO or DMF).
- a polar aprotic solvent e.g., DMSO or DMF.
- the aqueous medium does not contain organic solvents (e.g., alcohols such as Ci to C 6 alcohols) other than polar aprotic solvents at 10% or greater, 20% or greater, or 30% or greater.
- the aqueous medium does not contain alcohols at 1% or greater, 2% or greater, 3% or greater, 4% or greater, or 5% or greater.
- the aqueous medium does not contain any detectible alcohols.
- the reaction media of any of the steps of any of the methods disclosed herein consists essentially of water and, optionally, a polar aprotic solvent.
- the pH can be adjusted to a desired value or within a desired range.
- the pH the reaction mixture can be increased by addition of a base.
- suitable bases include ammonium hydroxide.
- nanoparticles or core-shell nanoparticles surface which can be surface functionalized with polyethylene glycol groups (i.e.,
- PEGylated comprises: a) forming a reaction mixture at room temperature (e.g., 15°C to 25°C depending on the location) comprising water and TMOS (a silica core forming monomer) (e.g., at a concentration of 1 ImM to 270mM), wherein the pH of the reaction mixture (which can be adjusted using a base such as, for example, ammonium hydroxide) is 6 to 9 (which results in formation of core precursor nanoparticles having an average size (e.g., longest dimension) of, for example, 1 nm to 2 nm); b) either i) holding the reaction mixture at a time (t 1 ) and temperature (T 1 ) (e.g., (t 1 ) 0.5 days to 7 days at room temperature to 95 °C (T 1 )), whereby nanoparticles (core nanoparticles) having an average size (e.g., longest dimension) of 2 to 15 nm are formed, or ii) cooling the reaction mixture to room
- the nanoparticles can be subjected to post-synthesis processing steps. For example, after synthesis (e.g., after e) in the example above) the solution is cooled to room temperature and then transferred into a dialysis membrane tube (e.g. a dialysis membrane tube having a Molecular Weight Cut off 10,000, which are commercially available (e.g., from Pierce)).
- a dialysis membrane tube e.g. a dialysis membrane tube having a Molecular Weight Cut off 10,000, which are commercially available (e.g., from Pierce)
- the solution in the dialysis tube is dialyzed in Dl-water (volume of water is 200 times more than the reaction volume, e.g. 2000ml water for a 10ml reaction) and the water is changed every day for one to six days to wash away remaining reagents, e.g. ammonium hydroxide and free silane molecules.
- the particles are then filtered through a 200nm syringe filter (fisher brand) to remove aggregates or dust.
- additional purification processes including gel permeation chromatography and high-performance liquid chromatography, can be applied to the nanoparticles to further ensure the high purify of the synthesized particles (e.g., 1% or less unreacted reagents or aggregates).
- the purified nanoparticles can be transferred back to deionized water if other solvent is used in the additional processes.
- the cores can be silicon cores.
- the reaction mixture used in silicon core formation can comprise TMOS as the only silicon core forming monomer.
- the cores can be aluminosilicate cores.
- the reaction mixture used in aluminosilicate core formation can comprise TMOS as the only silicon core forming monomer and one or more alumina core forming monomer (e.g., an aluminum alkoxide such as, for example, aluminum-tri-sec-butoxide or a combination of aluminum alkoxides).
- the pH of the reaction mixture is adjusted to a pH of 1 to 2 prior to addition of the alumina core forming monomer.
- the pH of the solution is adjusted to a pH of 7 to 9 and, optionally, PEG with molecular weight between 100 and 1,000 g/mol, including all integer values and ranges therebetween, at concentration of lOmM to 75mM, including all integer mM values and ranges therebetween, is added to the reaction mixture prior to adjusting the pH of the reaction mixture to a pH of 7 to 9.
- the reaction mixture used to form core nanoparticles can also comprise a dye precursor.
- the resulting core or core-shell nanoparticles have one or more dye molecules encapsulated or incorporated therein.
- core nanoparticle has 1, 2, 3, 4, 5, 6, or 7 dye molecules encapsulated therein.
- Mixtures of dye precursors can be used.
- the dye precursor is a dye conjugated to a silane.
- the dye can have an emission (e.g., fluorescence) wavelength of 400 nm (blue) to 800 nm (near-infrared).
- the dye is a NIR-dye.
- Suitable dyes include, but are not limited to, rhodamine green (RHG), tetramethylrhodamine (TMR), Cyanine 5 (Cy5), Cyanine 5.5 (Cy5.5), Cyanine 7 (Cy7), ATT0647N, Dyomics DY800, Dyomics DY782 and IRDye 800CW, and the nanoparticles surface functionalized with polyethylene glycol groups or the core-shell nanoparticles surface functionalized with polyethylene glycol groups have one or more fluorescent dye molecules encapsulated therein.
- RHG rhodamine green
- TMR tetramethylrhodamine
- Cyanine 5 Cyanine 5
- Cyanine 5.5 Cyanine 5.5
- Cyanine 7 Cyanine 7
- ATT0647N Dyomics DY800, Dyomics DY782 and IRDye 800CW
- a silica shell can be formed on the core nanoparticles.
- the silica shell is formed after, for example, core formation is complete.
- silica shell forming precursors include tetraalkylorthosilicates such as, for example, TEOS and TPOS. Mixtures of silica shell forming precursors can be used.
- TMOS is not a silica shell forming precursor.
- the silica shell forming precursor can be added to the reaction mixture as a solution in a polar aprotic solvent. Examples, of suitable polar aprotic solvents include DMSO and DMF.
- the shell forming monomer(s) is/are added in separate aliquots (e.g., 40 to 500 aliquots)
- the aliquots can include one or more shell forming precursor (e.g., TEOS and/or TPOS) and a polar aprotic solvent e.g., DMSO.
- Each aliquot can have 1 to 20 micromoles of shell forming monomer.
- the interval between aliquot addition can be 1 to 60 minutes, including all integer minute values and ranges therebetween.
- the pH of the reaction mixture can vary during the silica shell forming process. It is desirable to adjust the pH to maintain a pH of 7-8.
- the core or core-shell nanoparticles can by reacted with one or more PEG-silane conjugates.
- PEG-silane conjugates can be added together or in various orders. This process is also referred to herein as PEGylation.
- the conversion percentage of PEG-silane is between 5% and 40% and the polyethylene glycol surface density is 1.3 to 2.1 polyethylene glycol molecules per nm 2 .
- the conversion percentage of ligand-functionalized PEG-silane is 40% to 100% and the number of ligand-functionalized PEG-silane precursors reacted with each particle is 3 to 90.
- PEGylation can be carried out at a variety of times and temperatures.
- PEGylation can be carried out by contacting the nanoparticles at room temperature for 0.5 minutes to 24 hours (e.g., overnight).
- alumina-silicate nanoparticles e.g., alumina-silicate core nanoparticles or silica core silica shell nanoparticles
- the temperature is 80°C overnight.
- the chain length of the PEG moiety of the PEG-silane (i.e., the molecular weight of the PEG moiety) can be tuned from 3 to 24 ethylene glycol monomers (e.g., 3 to 6, 3 to 9, 6 to 9, 8 to 12, or 8 to 24 ethylene glycol monomers).
- the PEG chain length of PEG- silane can be selected to tune the thickness of the PEG layer surrounding the particle and the pharmaceutical kinetics profiles of the PEGylated particles.
- the PEG chain length of ligand- functionalized PEG-silane can be used to tune the accessibility of the ligand groups on the surface of the PEG layer of the particles resulting in varying binding and targeting performance.
- PEG-silane conjugates can comprise a ligand.
- the ligand is covalently bound to the PEG moiety of the PEG-silane conjugates (e.g., via though the hydroxy terminus of the PEG-silane conjugates).
- the ligand can be conjugated to a terminus of the PEG moiety opposite the terminus conjugated to the silane moiety.
- the PEG-silane conjugate can be formed using a heterobifunctional PEG compound (e.g., maleimido-functionalized heterobifunctional PEGs, NHS ester-functionalized heterobifunctional PEGs, amine- functionalized heterobifunctional PEGs, thiol-functionalized heterobifunctional PEGs, etc.).
- Suitable ligands include, but are not limited to, peptides (natural or synthetic), ligands comprising a radio label (e.g., 124 I, 131 1, 225 Ac or 177 Lu), antibodies, ligands comprising a reactive group (e.g., a reactive group that can be conjugated to a molecule such a drug molecule, gefitinib, etc.).
- a radio label e.g., 124 I, 131 1, 225 Ac or 177 Lu
- ligands comprising a reactive group e.g., a reactive group that can be conjugated to a molecule such a drug molecule, gefitinib, etc.
- PEG-silane conjugate comprising a ligand is added in addition to
- the conversion percentage of ligand- functionalized or reactive group-functionalized PEG-silane is 40% to 100% and the number of ligand-functionalized PEG-silane precursors reacted with each particle is 3 to 600.
- PEG-silane conjugate is added (e.g., in d) in the example above) a PEG-silane conjugate comprising a ligand (e.g., at concentration between 0.05 mM and 2.5 mM) is added at room temperature to the reaction mixture comprising the core nanoparticles or core-shell nanoparticles (e.g., from b) i) or b) ii), respectively, in the example above).
- a PEG-silane conjugate comprising a ligand e.g., at concentration between 0.05 mM and 2.5 mM
- the resulting reaction mixture is held at a time (t 4 ) and temperature (T 4 ) (e.g., (t 4 ) 0.5 minutes to 24 hours at room temperature (T 4 )), where at least a portion of the PEG-silane conjugate molecules are adsorbed on at least a portion of the surface of the core nanoparticles or core-shell nanoparticles (e.g., from b) in the example above).
- T 4 temperature
- reaction mixture is heated at a time (t 5 ) and temperature (T 5 ) (e.g., (t 5 ) 1 hour to 24 hours at 40 °C to 100 °C (T 5 )), where nanoparticles surface functionalized with polyethylene glycol groups comprising a ligand or core-shell nanoparticles surface functionalized with polyethylene glycol groups comprising a ligand are formed.
- T 5 time and temperature
- the concentration of PEG-silane no ligand is between lOmM and 75mM
- PEG-silane conjugate dissolved in a polar aprotic solvent such as, for example, DMSO or DMF
- T 6 temperature
- at least a portion of the PEG-silane conjugate molecules are adsorbed on at least a portion of the surface of the nanoparticles surface functionalized with polyethylene glycol groups comprising a ligand or at least a portion of the core-shell nanoparticles surface functional
- the PEG-silane has a reactive group on a terminus of the PEG moiety opposite the terminus conjugated to the silane moiety of the PEG-silane conjugate (is formed from a heterobifunctional PEG compound) and after formation of the nanoparticles surface functionalized with polyethylene glycol groups having a reactive group, and, optionally, polyethylene glycol groups, core-shell nanoparticles surface functionalized with polyethylene glycol groups having a reactive group.
- polyethylene glycol groups are reacted with a second ligand (which can be the same or different than the ligand of the nanoparticles surface functionalized with polyethylene glycol groups and polyethylene glycol group comprising a ligand or the core-shell nanoparticles surface functionalized with polyethylene glycol groups and polyethylene groups comprising a ligand) functionalized with a second reactive group (which can be the same or different than the reactive group of the nanoparticles surface functionalized with polyethylene glycol groups and polyethylene glycol group comprising a ligand or the core-shell nanoparticles surface functionalized with polyethylene glycol groups and polyethylene groups comprising a ligand) thereby forming nanoparticles surface functionalized with polyethylene groups functionalized with a second ligand and, optionally, polyethylene glycol groups, core-shell nanoparticles surface functionalized with polyethylene groups functionalized with a second ligand and polyethylene glycol groups and, optionally, polyethylene glycol groups.
- the PEG-silane has a reactive group on a terminus of the PEG moiety opposite the terminus conjugated to the silane moiety of the PEG-silane conjugate (is formed from a heterobifunctional PEG compound) and after formation of the nanoparticles surface functionalized with polyethylene glycol groups and, optionally having a reactive group, and, optionally, polyethylene glycol groups, core-shell nanoparticles surface functionalized with polyethylene glycol groups having a reactive group, and, optionally, polyethylene glycol groups, are reacted with a second ligand (which can be the same or different than the ligand of the nanoparticles surface functionalized with polyethylene glycol groups and polyethylene glycol group comprising a ligand or the core- shell nanoparticles surface functionalized with polyethylene glycol groups and polyethylene groups comprising a ligand) functionalized with a second reactive group (which can be the same or different than the reactive group of the nanoparticle
- the nanoparticles with PEG groups functionalized with reactive groups can be further functionalized with one or more ligands.
- a functionalized ligand can be reacted with a reactive group of a PEG group.
- suitable reaction chemistries and conditions for post-nanoparticle synthesis functionalization are known in the art.
- the nanoparticles can have a narrow size distribution.
- the nanoparticle size distribution (before or after PEGylation), not including extraneous materials such as, for example, unreacted reagents, dust particles/aggregates, is +/- 5, 10, 15, or 20% of the average particle size (e.g., longest dimension).
- the particle size can be determined by methods known in the art. For example, the particle size is determined by TEM, GPS, or DLS. DLS contains systematic deviation and, therefore, the DLS size distribution may not correlate with the size distribution determined by TEM or GPS.
- compositions comprising nanoparticles of the present disclosure.
- the compositions can comprise one or more types (e.g., having different average size and/or one or more different compositional feature).
- a composition comprises a plurality of core and/or core-shell nanoparticles (e.g., silica core nanoparticles, silica core-shell nanoparticles, aluminosilicate core nanoparticles, aluminosilicate core-shell nanoparticles.
- core and/or core-shell nanoparticles e.g., silica core nanoparticles, silica core-shell nanoparticles, aluminosilicate core nanoparticles, aluminosilicate core-shell nanoparticles.
- Any of the nanoparticles may be surface functionalized with one or more type of polyethylene glycol groups (e.g.,
- any of the nanoparticles can have a dye or combination of dyes (e.g., a NIR dye) encapsulated therein.
- the dye molecules are covalently bound to the nanoparticles.
- the nanoparticles can be made by a method of the present disclosure.
- the nanoparticles in a composition can have a variety of sizes.
- the nanoparticles can have a core size of 2 to 15 nm, including all 0.1 nm values and ranges therebetween.
- the nanoparticles have a core size of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 nm.
- at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% 99.9%, or 100% of the core and/or core-shell nanoparticles have a size (e.g., longest dimension) of 2 to 15 nm.
- At least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% 99.9%, or 100% of the core-shell nanoparticles have a size (e.g., longest dimension) of 2 to 50 nm. In various examples, at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the core and/or core of the core-shell nanoparticles have a size (e.g., longest dimension) of 2 to 15 nm.
- the composition may not be subjected to any particle- size discriminating (particle size selection/removal) processes (e.g., filtration, dialysis, chromatography (e.g., GPC), centrifugation, etc.).
- particle- size discriminating processes e.g., filtration, dialysis, chromatography (e.g., GPC), centrifugation, etc.
- the nanoparticles of the present disclosure are the only nanoparticles in the composition.
- composition can comprise additional components.
- the composition can also comprise a buffer suitable for administration to an individual (e.g., a mammal such as, for example, a human).
- the buffer may be a pharmaceutically-acceptable carrier.
- compositions as synthesized and before any post-synthesis
- processing/treatment can have nanoparticles, particles (2-15 nm), dust parti cles/aggregates (>20nm), unreacted reagents ( ⁇ 2 nm).
- the present disclosure provides uses of the nanoparticles and compositions of the present disclosure.
- nanoparticles or a composition comprising the nanoparticles are used in delivery and/or imaging methods.
- the ligands carried by the nanoparticles can include diagnostic and/or therapeutic agents (e.g., drugs).
- therapeutic agents include, but are not limited to, chemotherapeutic agents, antibiotics, antifungal agents, antiparasitic agents, antiviral agents, and combinations thereof.
- An affinity ligand may be also be conjugated to the nanoparticle to allow targeted delivery of the nanoparticles.
- the nanoparticle may be conjugated to a ligand which is capable of binding to a cellular component (e.g., on the cell membrane or in the intracellular compartment) associated with a specific cell type.
- the targeted molecule can be a tumor marker or a molecule in a signaling pathway.
- the ligand can have specific binding affinity to certain cell types, such as, for example, tumor cells.
- the ligand may be used for guiding the nanoparticles to specific areas, such as, for example, liver, spleen, brain or the like. Imaging can be used to determine the location of the nanoparticles in an individual.
- the nanoparticles or compositions comprising nanoparticles can be administered to individuals for example, in pharmaceutically-acceptable carriers, which facilitate transporting the nanoparticles from one organ or portion of the body to another organ or portion of the body. Examples of individuals include animals such as human and non-human animals. Examples of individuals also include mammals.
- Pharmaceutically acceptable carriers are generally aqueous based.
- materials which can be used in pharmaceutically-acceptable carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid;
- compositions comprising the present nanoparticles can be administered to an individual by any suitable route - either alone or as in combination with other agents.
- Administration can be accomplished by any means, such as, for example, by parenteral, mucosal, pulmonary, topical, catheter-based, or oral means of delivery.
- Parenteral delivery can include, for example, subcutaneous, intravenous, intramuscular, intra-arterial, and injection into the tissue of an organ.
- Mucosal delivery can include, for example, intranasal delivery.
- Pulmonary delivery can include inhalation of the agent.
- Catheter-based delivery can include delivery by iontophoretic catheter-based delivery.
- Oral delivery can include delivery of an enteric coated pill, or administration of a liquid by mouth.
- Transdermal delivery can include delivery via the use of dermal patches.
- nanoparticles, the path, location, and clearance of the NPs can be monitored using one or more imaging techniques.
- imaging techniques include Artemis
- This disclosure provides a method for imaging biological material such as cells, extracellular components, or tissues comprising contacting the biological material with nanoparticles comprising one or more dyes, or compositions comprising the nanoparticles; directing excitation electromagnetic (e/m) radiation, such as light, on to the tissues or cells thereby exciting the dye molecules; detecting e/m radiation emitted by the excited dye molecules; and capturing and processing the detected e/m radiation to provide one or more images of the biological material.
- e/m radiation such as light
- fiber optical instruments can be used.
- a method for imaging of a region within an individual comprises
- fluorescent particles are brighter than free dye, fluorescent particles can be used for tissue imaging, as well as to image the metastasis tumor. Additionally or alternatively, radioisotopes can be further attached to the ligand groups (e.g., tyrosine residue or chelator) of the ligand-functionalized particles or to the silica matrix of the PEGylated particles without specific ligand functionalization for photoinduced electron transfer imaging. If the radioisotopes are chosen to be therapeutic, such as 225 Ac or 177 Lu, this in turn would result in particles with additional radiotherapeutic properties.
- ligand groups e.g., tyrosine residue or chelator
- drug-linker conjugate where the linker group can be specifically cleaved by enzyme or acid condition in tumor for drug release
- drug-linker-thiol conjugates can be attached to maleimido-PEG-particles through thiol-maleimido conjugation reaction post the synthesis of maleimido-PEG-particles.
- both drug-linker conjugate and cancer targeting peptides can be attached to the particle surface for drug delivery specifically to tumor.
- the steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods and produce the compositions of the present disclosure.
- the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.
- compositions of the present disclosure are described:
- a method of making nanoparticles optionally surface functionalized with polyethylene glycol (PEG) groups (i.e., PEGylated), or core-shell nanoparticles optionally surface functionalized with PEG groups comprising a) forming a reaction mixture at room
- TMOS silica core forming monomer
- the pH of the reaction mixture (which can be adjusted using a base such as, for example, ammonium hydroxide) is 6 to 9 (which results in formation of core precursor nanoparticles having an average size (e.g., longest dimension) of, for example, 1 nm to 2 nm); b) either i) holding the reaction mixture at a time (t 1 ) and temperature (T 1 ) (e.g., (t 1 ) 0.1 hour to 7 days at room temperature to 95°C (T 1 ), such as 10-15 minutes), whereby nanoparticles (core nanoparticles) having an average size (e.g., longest dimension) of 2 to 15 nm are formed, or ii) cooling the reaction mixture to room temperature, if necessary, and adding a shell forming monomer (
- T 3 temperature (e.g., (t 3 ) 1 hour to 24 hours at 40 °C to 100 °C (T 3 )), whereby the nanoparticles surface functionalized with PEG groups or the core-shell nanoparticles surface functionalized with PEG groups are formed.
- reaction mixture further comprises alumina or aluminasilicate core forming monomer (e.g., aluminum alkoxides such as, for example, aluminum-tri-sec-butoxide) and the pH of the reaction mixture is adjusted to a pH of 1 to 2 prior to addition of the alumina or aluminasilicate core forming monomer and, optionally, PEG (e.g., PEG with molecular weight between 0. Ik and lk and concentration between lOmM and 75mM is added to the reaction mixture prior to adjusting the pH to a pH of 7 to 9, and the core is an aluminosilicate core.
- alumina or aluminasilicate core forming monomer e.g., aluminum alkoxides such as, for example, aluminum-tri-sec-butoxide
- PEG e.g., PEG with molecular weight between 0. Ik and lk and concentration between lOmM and 75mM is added to the reaction mixture prior to adjusting the pH to a
- a dye precursor e.g., a silane-dye conjugate such as a silane-NIR dye conjugate
- the nanoparticles surface functionalized with PEG groups or the core-shell nanoparticles surface functionalized with PEG groups have one or more fluorescent dye molecules covalently encapsulated therein.
- 1 to 7 an average of 1 to 7, e.g., 2) dye molecules (e.g., fluorescent dye molecules such as, for example, NIR dye molecules) are present in each of the nanoparticles surface functionalized with PEG groups or core-shell nanoparticles surface functionalized with PEG groups.
- dye molecules e.g., fluorescent dye molecules such as, for example, NIR dye molecules
- dye molecules e.g., fluorescent dye molecules such as, for example, NIR dye molecules
- the PEG-silane conjugate comprises a ligand (e.g., a peptide (natural or synthetic), a ligand comprising a moiety comprising a radio label (e.g., 124 I, 131 1, 225 Ac or 177 Lu), antibody, a ligand comprising a reactive group (e.g., a reactive group that can be conjugated to a molecule such a drug molecule, e.g., ) conjugated to a terminus of the PEG moiety opposite the terminus conjugated to the silane moiety (can be formed using a heterobifunctional PEG compound).
- a ligand e.g., a peptide (natural or synthetic)
- a ligand comprising a moiety comprising a radio label e.g., 124 I, 131 1, 225 Ac or 177 Lu
- a ligand comprising a reactive group e.g., a reactive group that can be conjugated to a molecule such
- a PEG-silane conjugate comprising a ligand e.g., at concentration between 0.05 mM and 2.5 mM
- PEG-silane conjugate comprising a ligand dissolved in a polar aprotic solvent such as, for example, DMSO or DMF is added at room temperature to the reaction mixture comprising the core nanoparticles or core-shell nanoparticles from b) i) or b) ii), respectively, holding the resulting reaction mixture at a time (t 4 ) and temperature (T 4 ) (e.g., (t 4 ) 0.5 minutes to 24 hours at room temperature (T 4 ))
- T 4 temperature
- at least a portion of the PEG-silane conjugate molecules are adsorbed on at least a portion of the surface of the core nanoparticles or core-shell nanoparticles from b
- heterobifunctional PEG compound and after formation of the nanoparticles surface functionalized with PEG groups having a reactive group, and, optionally, PEG groups, core- shell nanoparticles surface functionalized with PEG groups having a reactive group, and, optionally, PEG groups, are reacted with a second ligand (which can be the same or different than the ligand of the nanoparticles surface functionalized with PEG groups and PEG group comprising a ligand or the core-shell nanoparticles surface functionalized with PEG groups and polyethylene groups comprising a ligand) functionalized with a second reactive group (which can be the same or different than the reactive group of the nanoparticles surface functionalized with PEG groups and PEG group comprising a ligand or the core-shell nanoparticles surface functionalized with PEG groups and polyethylene groups comprising a ligand) thereby forming nanoparticles surface functionalized with polyethylene groups functionalized with a second ligand and, optionally, PEG groups, core-shell nanoparticle
- nanoparticles surface functionalized with PEG groups having a reactive group, and, optionally, PEG groups are reacted with a second ligand (which can be the same or different than the ligand of the nanoparticles surface functionalized with PEG groups and PEG group comprising a ligand or the core-shell nanoparticles surface functionalized with PEG groups and polyethylene groups comprising a ligand) functionalized with a second reactive group (which can be the same or different than the reactive group of the nanoparticles surface functionalized with PEG groups and PEG group comprising a ligand or the core-shell nanoparticles surface functionalized with PEG groups and polyethylene groups comprising a ligand) thereby forming nanoparticles surface functionalized with polyethylene groups functionalized with a second ligand and, optionally, PEG groups, core-shell nanoparticles surface functionalized with polyethylene groups functionalized with a second ligand and PEG groups and, optionally, PEG groups.
- the PEG-silane has a reactive group on a terminus of the PEG moiety opposite the terminus conjugated to the silane moiety of the PEG-silane conjugate (is formed from a heterobifunctional PEG compound) and after formation of the nanoparticles surface functionalized with PEG groups having a reactive group, core-shell nanoparticles surface functionalized with PEG groups having a reactive group, nanoparticles surface functionalized with PEG groups having a reactive group and PEG groups comprising a ligand, or core-shell nanoparticles surface functionalized with PEG groups having a reactive group and polyethylene groups comprising a ligand the reactive group are reacted with a second ligand functionalized with a reactive group (which can be the same or different than the ligand of the nanoparticles surface functionalized with PEG groups and PEG group comprising a ligand, or core-shell nanoparticles surface functionalized with PEG groups and polyethylene groups comprising a ligand)
- a composition comprising a plurality of core or core-shell nanoparticles surface functionalized with PEG groups or core-shell nanoparticles surface functionalized with PEG groups, wherein at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the core nanoparticles have a size (e.g., longest dimension) of 2 to 15 nm or core-shell nanoparticles have a core size (e.g., longest dimension) of 2 to 15 nm and/or a size (e.g., longest dimension) of 2 to 50 nm and the composition has not been subjected to any particle-size discriminating (particle size selection/removal) processes (e.g., filtration, dialysis, or centrifugation).
- particle-size discriminating particle size selection/removal
- the core is an aluminosilicate core.
- composition of Statement 12 wherein the PEG chain length of ligand-PEG-silane is longer than the PEG chain length of PEG-silane thereby ensuring accessibility of the functional ligands (e.g., if a PEG-silane with 6-9 EO monomers is used, the PEG chain of the ligand-PEG-silane has at least 12 EO monomers or if a PEG-silane with 12 EO monomers is used, the PEG chain of the ligand-PEG-silane has at least 16 EO monomers).
- the PEG chain length of ligand-PEG-silane is longer than the PEG chain length of PEG-silane thereby ensuring accessibility of the functional ligands (e.g., if a PEG-silane with 6-9 EO monomers is used, the PEG chain of the ligand-PEG-silane has at least 12 EO monomers or if a PEG-silane with 12 EO
- composition of Statement 12 further comprising radioisotopes attached to the ligand groups (e.g., tyrosine residue or chelator of the ligand-functionalized particles or to the silica matrix of the PEGylated particles without specific ligand functionalization), for photoinduced electron transfer imaging.
- radioisotopes attached to the ligand groups e.g., tyrosine residue or chelator of the ligand-functionalized particles or to the silica matrix of the PEGylated particles without specific ligand functionalization
- the radioisotopes are selected to be therapeutic, (e.g., 225 Ac or 177 Lu), thereby forming nanoparticles with radiotherapeutic properties.
- composition of Statement 12 wherein the composition comprises a drug-linker conjugate covalently attached to the functional ligands on the nanoparticles for drug delivery, wherein the linker group is configured to be cleaved by enzyme or acid condition in tumor for drug release.
- a method for imaging of a region within an individual comprising: (a) administering to the individual the composition of one of Statements 13-23 of the present disclosure, wherein the nanoparticles comprise one or more dye molecules; (b) directing excitation
- electromagnetic radiation e.g., light
- electromagnetic radiation into the subject, thereby exciting at least one of the one or more dye molecules
- detecting excited electromagnetic radiation e.g., light
- processing signals corresponding to the detected electromagnetic radiation to provide one or more images (e.g. a real-time video stream) of the region within the subject.
- Ultrasmall fluorescent silica nanoparticles (SNPs) and core-shell SNPs surface functionalized with polyethylene glycol (PEG), specific surface ligands, and overall SNP size in the regime below 10 nm are of rapidly increasing interest for clinical applications due to their favorable biodistribution and safety profiles.
- PEG polyethylene glycol
- specific surface ligands specific surface ligands
- overall SNP size in the regime below 10 nm are of rapidly increasing interest for clinical applications due to their favorable biodistribution and safety profiles.
- an aqueous synthesis methodology for the preparation of narrowly size-dispersed SNPs and core-shell SNPs with size control below 1 nm (e.g., at the level of a single atomic layer) is presented.
- Different types of fluorophores, including near infrared (NIR) emitters, can be covalently encapsulated.
- Brightness can be enhanced via addition of extra silica shells. This methodology further enables synthesis of ⁇ 10 nm sized fluorescent core and core-shell SNPs with previously unknown compositions.
- an aluminum sol-gel precursor leads to fluorescent aluminosilicate nanoparticles (ASNPs) and core-shell ASNPs.
- ASNPs fluorescent aluminosilicate nanoparticles
- Encapsulation efficiency and brightness of highly negatively charged NIR fluorophores is enhanced relative to the corresponding SNPs without aluminum. Resulting particles show quantum yields of -0.8 (e.g., starting to approach the theoretical brightness limit). All particles may be
- heterobifunctional PEGs can be employed to introduce ligands onto the PEGylated particle surface of fluorescent SNPs, core-shell SNPS, and their aluminum containing analogues, producing ligand functionalized ⁇ 10 nm NIR fluorescent nanoprobes.
- the SNPs and ASNPs described here and synthesized in water will be referred to as Cornell prime dots or C dots and A1C dots.
- These organic- inorganic hybrid nanomaterials may find applications in nanomedicine, including cancer diagnostics and therapy (theranostics).
- the water based synthesis approach is quite versatile and enables previously unknown inorganic compositions of the particles, without loss of particle size control.
- silica and mixed compositions derived from the addition of aluminum alkoxides as sol-gel precursors are investigated.
- the resulting growth conditions of these mixed inorganic Ps allow for more efficient incorporation of highly negatively charged NIR emitting fluorophores as compared to the plain silica based particles.
- the resulting NPs show enhanced quantum efficiency of encapsulated dye as compared to particles synthesized without the aluminum alkoxide addition.
- These aluminum containing fluorescent SNPs will be referred to as A1C dots.
- Fluorescent SNPs, core-shell SNPs, and their aluminum containing analogues are PEGylated to provide steric stability.
- heterobifunctional PEGs are employed to introduce ligands onto the PEGylated particle surface of fluorescent SNPs and core-shell SNPS, as well as their aluminum containing analogues, producing ligand functionalized ⁇ 10 nm NIR fluorescent nanoprobes for preclinical and clinical use in diagnostic and therapeutic applications. This is
- Aminopropyl)triethoxysilane APTES
- TMOS tetramethyl orthosilicate
- TEOS tetraethyl orthosilicate
- PEG polyethylene glycol chains
- Aldrich Aldrich
- Methoxy-terminated poly(ethylene glycol) chains PEG-silane, molar mass around 500
- Heterobifunctional PEGs with maleimide and NHS ester groups mal-PEG-NHS, molar mass around 800
- Acetic acid is purchased from Mallinckrodt.
- Cy5 and Cy5.5 fl orescent dyes are purchased from GE.
- Rhodamine green (RhG) and tetramethylrhodamine (TMR) fluorescent dyes are purchased from Life Technologies.
- DY782 florescent dye is purchased from Dyomics and CW800 florescent dye is purchased from Li-cor. Absolute anhydrous 99.5% ethanol is purchased from Pharmco-Aaper.
- Cyclo (Arg-Gly-Asp-D-Tyr-Cys) peptide, c(RGDyC) is purchased from Peptide International.
- Deionized water (DI water) is generated using a Millipore Milli-Q system.
- reaction conditions are listed as occurring overnight, the time the reaction can occur can range from about 8 hours to about 24 hours or even greater.
- the resulting particle solution is then subjected to long term storage at room temperature and characterization including TEM, DLS, TGA and NMR.
- the molar ratios of the reaction are 1 TMOS : 0.093 ammonia : 0.49 PEG-silane : 1292 H 2 0.
- Particles size was varied by tuning synthesis conditions. Details are summarized in Table 1.
- the same particles with the same size dispersity and structure control can also be synthesized using a 27wt% ammonium hydroxide solution instead of the 2.0M ammonia in ethanol as the ammonium hydroxide source as long as the solution pH is tuned to around 8.
- the silane-conjugated fluorophore is then added together with TMOS into the synthesis solution to co-condense into the particles.
- the molar ratio of silane-conjugated fluorophore to TMOS is around 1 : 1000.
- the remainder of the synthesis protocol is the same as described for the synthesis of the 4.2nm particles under section 2.2.
- any fluorophore-labeled particles prepared in this study are further purified by GPC after the filtration step to further remove any remaining free dye molecules, which could disturb FCS and emission spectra measurements, and to maximize the fluorescent particle product purity.
- the cleaned particle solution is concentrated by about 30 times using spin-filters (GE healthcare Vivaspin with MWCF 30k) and then purified by GPC column (see section 2.8 for details). Since the solvent used in the GPC setup is a 0.9wt% NaCl solution, the purified particles are finally transferred back to DI water using spin-filters for further characterizations and long-term storage.
- the purified particles are first concentrated by 30 times using spin- filters (GE healthcare Vivaspin with MWCF 30k). DI water is then added into the
- concentrated particle solution to dilute it back to the normal volume. This process is repeated for at least 8 times to decrease the concentration of NaCl to close to zero.
- the purified particle sample is then subjected to long-term storage at 40°C and for further
- the solution is then diluted 5 times with DI water. After that, a mixture of TEOS and DMSO (volume ratio 1 :4) is dosed into the solution under vigorous stirring at room temperature. The volume of each dose is lOul and the time gap between doses is 30 minutes. 50 doses are added for the addition of one layer of silica shell resulting in a shell thickness close to 0.5nm (particle size increase by around 1 nm). This process is repeated until the desired layers of shells (e.g., 1-4) are added. During the shell addition, the solution pH decreases as the result of the addition of extra TEOS and the formation of silicic acid.
- a mixture of TEOS and DMSO volume ratio 1 :4
- the volume of each dose is lOul and the time gap between doses is 30 minutes. 50 doses are added for the addition of one layer of silica shell resulting in a shell thickness close to 0.5nm (particle size increase by around 1 nm). This process is repeated until the desired layers of
- PEGylated aluminosilicate core-silica shell aluminosilicate nanoparticles For the synthesis of sub-10 nm PEGylated aluminosilicate nanoparticles (AS Ps), 1ml of 0.5N HC1 solution is added into 9 ml of DI water and the solution is stirred for 10 minutes. Following that, 0.43 mmol of TMOS and 0.043 mmol of aluminum-tri-sec-butoxide (dissolved in isopropanol with volume ratio 1 :9) are added under vigorous stirring at room temperature. 10-15 minutes later, 0.21 mmol PEG-silane is added followed by switching back of solution pH to neutral via adding about 140 ⁇ of 27 wt% ammonium hydroxide. The final pH is double checked with pH paper. Afterward, the solution is kept at 800°C overnight without stirring. The remainder of the synthesis protocol follows the same procedures as described for the synthesis of 4.2 nm particles in section 2.2.
- volume ratio 1 :8 is dosed into the solution under vigorous stirring at room temperature.
- the volume of each dose is 10 ⁇ and the time gap between doses is 30 minutes.
- 50 doses are added for the addition of one layer of silica shell resulting in a shell thickness close to 0.5nm (particle size increase by around lnm). This process is repeated until the desired layers of shells (e.g., 2) are added.
- 0.21 mmol PEG-silane is added and the 800°C heat treatment is applied without stirring.
- the remainder of the synthesis protocol follows the same procedures as described for the synthesis of 4.2 nm particles in section 2.2.
- the addition of PEG-silane before switching solution pH back to neutral is not necessary, but this can improve the monodispersity of synthesized ASNPs and prevent their aggregation during the process of changing pH.
- fluorophore is added right after the addition of TMOS and aluminum-tri-sec-butoxide using the same conjugation conditions and dye concentration as described in section 2.3.
- the remainder of the synthesis protocol is the same as described for the synthesis of blank ASNPs in section 2.5 and purification steps are applied as described above in section 2.3.
- the molar ratio of c(RGDyC) : NHS-PEG-mal : APTES is 1.1 : 1.0 : 0.9 to ensure every heterobifunctional PEG condensed on the particle surface has c(RGDyC) attached.
- the produced c(RGDyC) -PEG-silane is added followed by the addition of PEG-silane in the PEGylation step during nanoparticle synthesis.
- Different molar ratios of c(RGDyC) -PEG-silane : PEG- silane can be used to vary the amount of ligands on the particle surface.
- a molar ratio of c(RGDyC) -PEG-silane : PEG-silane of about 1 : 40 gives around 22 c(RGDyC) ligands per 7nm diameter particle, while decreasing the ratio to 1 :400 will result in about 5 c(RGDyC) ligands per 7nm particle.
- the remainder of the synthesis is the same as that described for the conventional PEGylation in section 2.
- the same methodology can be applied to all particles described in this study for producing surface functionalized probes, including blank and fluorescent SNPs, core-shell SNPS, and their aluminum containing analogues, all with different types of covalently encapsulated fluorophores.
- ligands that can be used in this way include, but are not limited to, other linear and cyclic peptides, antibody fragments, various DNA and RNA segments (e.g. siRNA), therapeutic molecules including drugs and radioisotopes and their respective chelating moieties, as well as combinations thereof.
- siRNA DNA and RNA segments
- therapeutic molecules including drugs and radioisotopes and their respective chelating moieties, as well as combinations thereof.
- characterization is performed using a BioLogic LP system equipped with a 275nm UV detector and with resin Superdex 200 from GE healthcare. While the blank S Ps can hardly be detected by the 275nm UV detector due to the low absorbance of silica, the fluorescent SNPs show strong signals in the GPC setup because the encapsulated fluorophores have absorbance overlapping with the 275nm detecting channel. As a result, GPC can be used to further increase the purity of cleaned C dot products for characterization and further clinical applications.
- the GPC system is calibrated by protein standards from Bio-Rad, which are a mixture of thyroglobulin, bovine ⁇ -globulin, chicken ovalbumin, equine myoglobin, and vitamin B12 with known molar masses. Afterwards, around 400 ⁇ of particle solution is injected into the GPC setup and fractions are collected by a BioFrac fraction collector. A detailed analysis of different GPC factions is displayed in Figure 15. By collecting the particle factions, the particle product purity can be further maximized.
- TEM Transmission electron microscopy
- FEI Tecnai T12 Spirit microscope operated at an acceleration voltage of 120kV.
- Hydrodynamic particle sizes and size distributions are measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-SZ operated at 200°C.
- DLS dynamic light scattering
- Each DLS sample is measured three times and results are superimposed in the respective figures in this paper.
- Number percentage curves are used to present the measurement results. The average diameter of each sample is calculated by averaging the mean diameters of number percentage curves from three measurements.
- FCS Fluorescent auto-correlation spectroscopy
- hydrodynamic size, brightness per particle and particle concentration are obtained from fits of the FCS auto-correlation curves. Dividing particle concentration obtained from FCS by fluorophore concentration obtained from absorbance measurements, the number of fluorophores per particle is calculated as described.
- the 27A1 NMR experiments are performed on a Bruker Avance NMR spectrometer with a 16.45T magnet (182.47 MHz 27A1 Larmor frequency) using a probe head for rotors of 2.5 mm diameter.
- Potassium alum serves as external 27A1 NMR chemical shift secondary reference (at -0.033 ppm) and for calibration of the 90 degree pulse lengths and if power.
- the final 27 Al NMR MAS spectra are acquired with a nominally 10 degree direct excitation pulse at 95 kHz rf field strength, adding up to 6144 scans with 100 ms repetition times, while spinning the sample at 15.00 kHz at the magic angle.
- the 27A1 background of the probe head and rotor are characterized by acquiring the spectrum of an empty rotor under identical conditions and subtracting it from the sample spectra.
- TGA Thermogravimetric analysis
- the particle solution is first frozen in liquid nitrogen and then left under vacuum at -200°C over three nights to dry.
- the powder after freeze-drying is further left under vacuum at 600°C overnight.
- the dried out particle sample is then subjected to TGA.
- the TGA is conducted using a TA Instruments Q500 thermogravimetric analyzer. During the measurement, the temperature is increased from room temperature to 1000°C with a ramp of 100°C/min and then remains at 1000°C for 2 h to fully exclude any residual water. Afterward, the temperature is further increased to 6000°C with a ramp of 100°C/min removing any organic moieties and leaving pure inorganic silica or aluminosilicate behind.
- a total of about 800 S1O2 units present inside the core particle are used, which agrees well with a calculation using the density of amorphous silica.
- a volume inside this core particle is manually created where one encapsulated Cy5 fluorophore is drawn.
- 16 c(RGDyC) -functionalized PEG chains covalently bonded to the silica particle surface are added manually.
- the final drawing represents one C dot particle with a ⁇ 3 nm core, -0.5 nm shell, one encapsulated Cy5 fluorophore, around 100 PEG chains, and 16 c(RGDyC) ligands on surface. It is important to note that the model is not the result of a true simulation, but is rather a scaled schematic drawing which provides a realistic visualization of the relative size scale of the different building blocks of one C dot particle.
- FIG. 1 presents the aqueous particle synthesis pathways pursued in this study together with the chemical and physical structures of the products.
- TMOS tetramethyl orthosilicate
- PEG-silane with molar mass around 500 g/mole is added into the reaction vessel to terminate particle growth.
- a subsequent heat treatment at elevated temperature (80°C) is then applied to enhance the condensation degree of PEG-silane on the particle surface.
- the synthesized particles are cleaned though dialysis to remove reaction reagents and then filtered by a 200 nm syringe filter to remove any aggregates or dust before further characterizations.
- the particle growth period is defined by the time window between the addition of TMOS and the addition of PEG-silane.
- particle PEGylation is a part of the synthesis, the particles are already surface modified with PEG chains once synthesized and are stable (e.g. in high salt containing buffer solutions).
- Particle size is controlled by varying reaction parameters including concentration of TMOS, concentration of ammonium hydroxide, length of particle growth period, and reaction temperature.
- Figure 2a-i display transmission electron microscopy (TEM) images at two different magnifications of particles grown under different conditions, revealing the size and size distribution control.
- Figure 2d shows 4.2nm diameter S Ps synthesized using the following parameters: room temperature, 0.043M TMOS, 0.002M ammonium hydroxide, and 12-24 hours growth period.
- the diameter of 4.2nm is determined by dynamic light scattering (DLS), while the particle diameter as obtained from TEM image analysis is only around 2-3nm.
- the particle size measured by DLS is slightly larger than that by TEM because DLS measures hydrodynamic size which includes PEG chains and hydration layers, while TEM only provides information about the diameter of the projected silica core.
- the exponential part represents the rapid particle formation kinetics at the beginning of the reaction and the linear part represents the kinetics of the subsequent controlled aggregation step.
- y is the average particle diameter at time t
- A is the relative amplitude of particle growth from the fast particle formation kinetics with characteristic time tR
- B is the particle growth rate of the later controlled aggregation process
- yo+A is the initial size of silica precursor. Since the FCS traces the Cy5-silane whose hydrodynamic size is about 1.2-1.5nm, the fitted values of yo+A are consistent with the expectation.
- FIG. 2j A comparison of size and size distributions as measured by DLS of the nine particle batches with different average diameters is displayed in Figure 2j.
- the data demonstrates precise particle size control with steps below 1 nm. Considering that a single S1O2 atomic layer is roughly around 0.4 nm thick, this is equivalent to controlling the particle growth at the level of the deposition of single atomic layers around the particles. Since this control is achieved by varying a combination of four reaction parameters, the synthesis system is endowed with a high degree of chemical versatility. This implies that narrowly size-dispersed ultrasmall S Ps can be synthesized under very different conditions, which should be compatible with other particle modification chemistries.
- tetraalkoxysilanes and particularly of TMOS, is greatly increased relative to alcohol, and thus even at close-to-neutral pH hydrolysis is still fast enough to generate a homogeneous silicic acid precursor solution.
- FCS fluorescence correlation spectroscopy
- FCS autocorrelation curves are fitted by the following equation:
- G(x) l+l/N- l/(l-A)-(l-A+A-e A (-x/xR ) ) ⁇ 1/(1+ ⁇ / ⁇ )* 1/V(1+S a 2-T/TD ),
- S is the structure factor (ratio between the shorter axis and the longer axis of the elliptical focal spot of FCS setup)
- N is the average number of particles present in the focal spot
- A is the amplitude of triplet state correction
- TR is the characteristic time of triplet state
- TD diffusion time.
- the structure factor S is obtained from the setup calibration and is fixed in the fit, while the values of N, A, TR and ID are obtained from the fit.
- Figure 10 shows an example of this type of curve fitting. Based on the fitted parameters, average hydrodynamic diameter of particles, fluorescent brightness per particle and concentration of fluorescent particles can be obtained.
- encapsulated Cy5 dye is brighter than free dye, and that encapsulated Cy5 dye brightness increases with particle size.
- This brightness enhancement has been correlated to an increase in quantum yield due to a change in dielectric constant moving from water to silica, as well as the increased rigidity of the dye in the silica environment.
- Cy5 fluorophores As overall particle size increases, Cy5 fluorophores have a higher probability of being well encapsulated by the silica matrix leading to the observed higher quantum enhancement.
- the number of fluorophores per particle as well as the brightness per particle can be obtained from FCS derived solution concentration in conjunction with absorbance data. The results are displayed in Figure 2m and 2n, respectively.
- each synthesized particle of the different synthesis batches contains around one to two fluorophores, and the number of fluorophores per particle slightly increases as particle size increases.
- one Cy5 fluorophore is around 1.2 nm in hydrodynamic diameter and has one negative charge (see molecular structure in Figure 1) repelling it from the negatively charged silica, an average of 1-2 fluorophores per particle is a reasonable number.
- the brightness per particle as measured by FCS directly is at least two times higher than the brightness per single Cy5 fluorophore in water, and increases as particle size increases.
- the brightness per particle can also be estimated from the product of the brightness per fluorophore and the number of fluorophores per particle (see black dots in Figure 2m). While the overall trends in behavior are the same, it is interesting to note that the brightness per particle as measured directly by FCS
- the silica source used in the shell additions is switched from TMOS to TEOS, because of its slower hydrolysis rate.
- the hydrolysis of TEOS can be tuned to be just a little bit slower than the time needed for a single does to thoroughly mix with the entire solution. In this way, once TEOS of each dose spreads out, it hydrolyzes and condenses onto the existing particles fairly fast. As a result, additional silica shells can be efficiently added without secondary particle nucleation, resulting in an increase of average particle size.
- the size of SNP cores can be tuned via controlled aggregation while extra silica shells can be added via monomer addition ( Figure 1).
- Figure 1 In order to show the principle that these two particle growth pathways can be switched in a single synthesis of fluorescent core-shell SNPs, a batch of particles is grown first through controlled aggregation by varying reaction temperature, then through monomer addition by further dosing extra silica source. Small amounts of particles are aliquoted for PEGylation at different synthesis moments and then characterized. Results show a consistent increase of particle size as well as of encapsulated dye quantum enhancement during the synthesis (for details see Figure 13). Results demonstrate that the architecture of ⁇ 10 nm fluorescent core-shell SNPs, via core diameter and shell thickness, can be tuned at the sub nanometer single atomic layer level by the particle growth conditions described herein.
- particle #3 PEGylated to generate particle #3.
- the diameter of particle #1 is 5.9 nm
- the diameter of particle #2 increases to 9 nm after heat treatment.
- This increase of particle size is due to the controlled aggregation of small particles at high temperature.
- This aggregation also increases the number of dyes per particle from 1.4 to 2.6.
- a thin silica shell is added onto the existing particle surface through monomer addition and the particle diameter further increases to 11.4 nm.
- particle #3 has an estimated core size slightly smaller than 9 nm and a silica shell with thickness around 1 nm. Since it has the same core size as particle #2, the number of dyes per particles remains about the same.
- the quantum enhancement of encapsulated dyes slightly increases from 1.7 to 1.8 due to the fact that the extra silica source further densifies the core matrix and the silica shells further protects the dyes inside the core.
- the number of dyes per particle could be increased by expanding the core size while the quantum efficiency could be enhanced by adding a thin layer of blank silica shell.
- the brightness of a ⁇ 10 nm particle could be optimized by precisely tuning both the core size and shell thickness through selecting the desired particle growth pathway.
- Table 3 Characterization results of three sets of particles quenched at different synthesis time points. Diameter and brightness are derived from FCS measurements while the # of dyes per particle is calculated from steady-state absorbance measurements and FCS concentration results.
- Figure 4g and 4h compare the absorbance/emission spectra and the solution appearance of the different C dots, respectively. It is interesting to note that the particle synthesis is compatible with different types of fluorophores with optical characteristics all the way from the blue part of the optical spectrum to the NIR. Especially in the 650-800 nm regime, different color NIR C dots can be synthesized enabling multi-color NIR imaging and NIR multiplexing.
- Table 4 Comparison of C dots from different types of fluorophores. Diameter and brightness are measured by FCS while the number of dyes per particle is calculated from steady-state absorbance measurements on different C dots and particle concentrations as measured by FCS.
- Fluorophores with emission spectra moving further out into the NIR typically have larger molar mass and size, therefore requiring more negatively charged sulfate groups on the periphery of their delocalized ⁇ -electron systems to generate the desired water solubility, e.g. compare molecular structures of Cy5 and Cy5.5 in Figure 1. Since silica above its isoelectric point at pH>2.7 is also negatively charged, it can be challenging to covalently encapsulate such highly negatively charged NIR fluorophores into SNPs due to increasing electrostatic repulsive interactions.
- the aqueous SNP growth solution pH is adjusted to values (pH ⁇ 1.5) slightly below the isoelectric point of silica (pH -2.7).
- Aluminum alkoxides are can be good choice for this purpose, as alumina derived from its alkoxides has already been approved for injection as one of the most common immunization adjuvants.
- the resulting ultrasmall NIR fluorescent aluminum containing SNPs may therefore also have potential for clinical translation.
- the solution pH is adjusted to around 1.5 using HC1.
- An aluminum-tri-sec-butoxide/isopropanol mixture and TMOS (molar ratio around 1 : 10) are then added simultaneously followed by addition of PEG-silane after 10 minutes of reaction time to terminate particle growth.
- an 80°C heat treatment is applied to enhance covalent particle PEGylation.
- TEM and DLS Figure 14
- silica nanoparticles are not stable at the strong acid condition.
- pH 2.7 the isoelectric point of silica
- the surface charge density of S Ps is not high enough to electrostatically stabilize particles and consequently particle aggregation occurs.
- a Cy5 doped PEGylated SNP synthesis batch is sorted into different factions by GPC and each faction is then subjected to FCS characterization.
- the average concentration of particles, particle brightness and particle diameter of each faction are shown in the upper three graphs in Figure 15.
- the GPC peak at around 18 minutes corresponds to objects in solution with a diameter around 1-2 nm, which in turn corresponds well to the size of free Cy5-silane conjugates or small self-condensed Cy5-silane conjugate clusters.
- the brightness per object corresponding to the 18 minute peak is close to the brightness of a single Cy5 fluorophore.
- This peak may be from free Cy5-silane dye conjugate or Cy5 conjugate clusters which are not encapsulated into particles.
- the main GPC peak at around 12 minutes corresponds to objects in solution with a diameter of around 6 nm and brightness levels more than two times the brightness of free Cy5 fluorophore. Both parameters correspond well to what is expected for the targeted fluorescent Cy5 encapsulating S Ps.
- the main peak at around 12 minute may be from the desired C dots.
- the GPC peak at around 6-7 minutes corresponds to objects in solution with a fairly big diameter of up to 20 nm. Interestingly, the brightness per object corresponding to this peak is similar to that of a single fluorescent S P. This peak should therefore not be assigned to objects coming purely from SNP aggregation.
- the peak at around 7 minutes stems from larger aggregates, which usually will be filtered away via cleaning.
- the peak at around 18 minutes is from unreacted dye molecules, which will be washed away through dialysis.
- the peak at around 12 minutes is the main particle peak.
- Heterobifunctional PEGs are employed to introduce easily accessible ligands onto the PEGylated particle surface of fluorescent SNPs and core-shell SNPS, as well as their aluminum containing analogues. This produces ligand functionalized ⁇ 10 nm NIR
- Ligands interesting for such applications include, but are not limited to, peptides, antibody fragments, various DNA and RNA segments (e.g. siRNA), therapeutic molecules including drugs as well as radioisotopes and their respective chelating moieties, and combinations thereof.
- siRNA DNA and RNA segments
- proof-of-principle is demonstrated using ⁇ ⁇ ⁇ 3 integrin- targeting cyclic(arginine-glycine-aspartic acid, tyrosine-cysteine) peptides, c(RGDyC), containing a tyrosine (Y) residue to bind a radioisotope, e.g.
- heterobifunctional PEGs with NHS ester and maleimide groups at their chain ends are first conjugated with amino silane through reaction of the amine with the NHS ester group.
- the resulting silane- PEG-maleimide is further conjugated with c(RGDyC) through thiol maleimide reaction to produce c(RGDyC) functionalized PEG-silane (c(RGDyC)-PEG-silane).
- heterobifunctional PEG is chosen to be a bit longer as compared to the PEG-silane (800 g/mole as compared to 500 g/mole, respectively). This ensures that the surface ligands are sticking out a bit beyond the surface of the PEG-silane coating and are therefore easily accessible (see also models in Figure 7).
- the final c(RGDyC)-PEG-silane is added into the nano-dot synthesis solution together with monofunctional PEG-silane in the PEGylation step. As a result, the surface of the synthesized nano-dots is covalently covered by both the common PEG chains and the longer c(RGDyC) functionalized PEG chains (Fig. 1).
- Figure 6b compares the absorbance spectra of free c(RGDyC) peptide, free Cy5 dye, and Cy5 encapsulated C dot cores (Cy5-C dots, no silica shell) with / without c(RGDyC) surface functionalization.
- the c(RGDyC) peptide has an absorbance peak at 275 nm from the tyrosine residue, while neither the Cy5 free dye nor the Cy5-C dots show detectable signals in this wavelength range. This absorbance peak can thus be used to verify successful particle surface functionalization.
- c(RGDyC) -PEG-Cy5-C dots show an absorbance peak at around 275 nm in addition to the 650 nm Cy5 absorbance peak. Furthermore, as shown in Figure 16, the FCS measured autocorrelation curve of c(RGDyC) -PEG-Cy5-C dots slightly shifts to the right compared to the dots without c(RGDyC) surface modification, indicating a ⁇ 1 nm particle size increase after attaching c(RGDyC) ligands to the particle surface (Table 6). These data suggest the c(RGDyC) peptides have been successfully attached to the particle surface.
- the number of c(RGDyC) ligands per C dot can be calculated. As shown in Table 6, from this synthesis batch on average around 16 c(RGDyC) peptides are attached to a single Cy5-C dot (no silica shell). End ligand numbers can be varied via the synthesis conditions, e.g. by changing the ratio of ligand-bearing and plain PEG-silane added in the particle synthesis step. Table 6. Characterization of various c(RGDyC) functionalized C dots, including fluorescent S Ps (C dots, core only), core-shell S PS (core-shell-C'dot), and their aluminum containing analo;
- the number of c(RGDyC) ligands per C dot can be estimated in a similar way as the calculation of the number of dyes per C dot.
- the height of the 275nm c(RGDyC) peak in the c(RGDyC)-C dot optical spectrum ( Figure 6b) can be estimated through subtracting from it the absorbance spectrum background obtained from non-c(RGDyC) ligand containing C dots. Then, by dividing the resulting c(RGDyC) peak height by the extinction coefficient of tyrosine, e.g.
- the concentration of total number of c(RGDyC) ligands present in the solution can be calculated. Meanwhile, the concentration of C dots in solution can be measured by FCS. Finally, by taking the ratio of c(RGDyC) concentration to C dot particle concentration the number of c(RGDyC) ligands per particle can be estimated. The results suggest that the number of c(RGDyC) ligands on the various C dots synthesized in this study is between 12 and 22 (see Table 6).
- c(RGDyC) peptides are attached to types of C dots under similar reaction conditions.
- these include C dots with additional silica shells, with different types of fluorophore (Cy5.5 vs. Cy5) and with different composition (A1C dots vs. C dots).
- all these synthesized particles show the absorbance peak at around 275 nm characteristic for c(RGDyC) indicating successful c(RGDyC) surface modification in all cases.
- the number of c(RGDyC) peptides per particle as derived from these optical measurements combined with FCS results slightly varies from 12 to 22 (Table 6).
- ligand density is another parameter that is expected to control biological response, and can be tuned via the synthesis parameters discussed in this study.
- the surface density of monofunctional PEG chains and c(RGDyC) -functionalized PEG chains of the synthesized ⁇ 7 nm C dot are estimated to be roughly 1.7/nm 2 and 0.2/nm 2 , respectively.
- the overall surface density of PEG chains on a high curvature C dot surface is very close to the reported ligand densities of shorter functional alkyl-silane monolayers on planar silica which is between 1.2/nm 2 and 2.2/nm 2 , or between 0.83 nm 2 and 0.45 nm 2 when expressed as area per head group.
- a TGA curve for a 6 nm blank non-fluorescent PEGylated S P (without fluorophore encapsulation) is displayed in Figure 17.
- the 6 nm average size is the hydrodynamic diameter as measured by DLS.
- the weight loss up to temperatures of 600°C indicates the presence of organic content in the particle sample. Since this sample synthesis did not include encapsulation steps of a fluorescent dye, the only organic content of the particle stems from the PEG chains on the particle surface.
- the TGA curve becomes flat at around 600°C suggesting most of the organic content has been burned off leaving inorganic silica as the remaining constituent. From this data it can then be estimated that the weight ratio between silica and PEGs is around 52%:48%.
- the number of PEG chains per particle can be estimated by assuming a silica density of about 1.9-2.2 g/cm 3 . Results suggest for this simple S P there are around 80-100 PEG chains on the particle surface. This translates to a PEG density on the particle surface of about 1.7 chains/nm 2 , which is equivalent to an area per PEG-silane head group of around 0.6 nm 2 . The number of PEGs per particle goes up as particle size increases.
- the models in Figure 7b and 7c represent a cut through a C dot with a ⁇ 3 nm diameter silica core encapsulating one Cy5 fluorophore (Figure 7b, top inset), a -0.5 nm thick silica shell, a -1.5 nm thick PEG layer, and 16 c(RGDyC) ligands ( Figure 7b, bottom inset) at the end of heterobifunctional PEGs that are a bit longer than the PEG-silanes.
- this silica nanoparticle consists of about 800 SiCh units and about 100 PEG chains on the particle surface.
- the overall particle size is around 7.5 nm and the overall particle molar mass is about 110 kDa.
- the length of a Cy5 fluorophore is between 2 and 3 nm although its hydrodynamic diameter as measured by FCS is only slightly larger than lnm (Table 3). Based on Figure 7, it can be concluded that a location of the covalently bonded Cy5 fluorophore exactly inside the 3nm silica core is unlikely given the stochastic nature of the encapsulation process and the electrostatic repulsion between Cy5 and negatively charged deprotonated silica surface hydroxyl groups.
- fluorophores including NIR emitting dyes can be encapsulated into the particles to produce fluorescent probes whose brightness can be further enhanced via addition of extra silica shells before PEGylation.
- This methodology further enables synthesis of ⁇ 10 nm sized fluorescent SNPs with other compositions.
- an aluminum sol-gel precursor leads to aluminum containing fluorescent core and core-shell nanoparticles, for which not only the encapsulation efficiency of highly negatively charged NIR fluorophores is enhanced relative to the silica particles, but also the quantum enhancement of individual fluorophores is starting to approach the theoretical brightness limit.
- heterobifunctional PEGs can be employed to introduce easily accessible ligands onto the PEGylated particle surface of fluorescent SNPs and core- shell SNPS, as well as their aluminum containing analogues, producing ⁇ 10 nm NIR fluorescent nanoprobes for preclinical and clinical use in diagnostic and therapeutic applications.
- NP structure correlations as described here may also help improve fundamental understanding of the mechanisms of early growth states of SNPs.
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JP2018519261A (en) | 2018-07-19 |
KR20180033126A (en) | 2018-04-02 |
CN107835696A (en) | 2018-03-23 |
BR112017023718A2 (en) | 2018-07-17 |
AU2016257431A1 (en) | 2017-12-21 |
JP2023055854A (en) | 2023-04-18 |
EP3291840A4 (en) | 2019-01-02 |
CN107835696B (en) | 2024-06-04 |
JP6908276B2 (en) | 2021-07-21 |
JP2021152070A (en) | 2021-09-30 |
BR112017023718B1 (en) | 2023-11-14 |
US20180133346A1 (en) | 2018-05-17 |
JP7458671B2 (en) | 2024-04-01 |
EP3291840A1 (en) | 2018-03-14 |
US12115231B2 (en) | 2024-10-15 |
AU2016257431B2 (en) | 2021-08-05 |
US11291737B2 (en) | 2022-04-05 |
US20230140770A1 (en) | 2023-05-04 |
CA2985083A1 (en) | 2016-11-10 |
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