Patent
US 10,994,016Graphene Oxide Cellular Delivery of Hydrophilic Small Molecules
Mark C. Hersam
US
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Patent
Atlas literature
Patent
US 10,994,016Patent drawings and their descriptions. Click a drawing to enlarge it.
Figure 1. The cellular delivery of bioactive molecules can be enhanced by simple mixing and co-incubation with graphene oxide in culture. [0
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 4. Structures of the 14 molecules tested with GO for adsorption, sedimentation, and cellular delivery. The quenching properties of GO usually hinder quantitative studies. The challenge is overcome by using Gd(III) to enable analytical quantitation by ICP. 34724791 [0
Figures 5A-C. A) GO adsorbed a(variety of molecules in cell culture media with amines 8, 9, 10, 12) producing the strongest interaction and(hydroxyls 2) and(phosphates 3) placing a far second. B) GO sedimentation induced by molecular adsorption was measured by the fraction of GO remaining in the …
Figures 6A-C. Adsorption as a function of A) charge, B) hydrophobicity, and C) hydrogen bonding potential of the adsorbing molecule on GO. No significant correlation was observed between adsorption and any of the chemical variables, indicating that GO adsorption in media cannot be simply predicted …
Figures 7A-C. Molecular adsorption, GO sedimentation, and GO size control the cellular delivery process. Molecules that A) exhibited higher adsorption and B) induced increased sedimentation experienced greater delivery enhancement by GO. Simple linear regression required separate fits for the two GO …
Figure 8. A significant correlation between delivery enhancement and adsorption can be found for molecules that did not increase GO sedimentation. The correlation was found for both the 150 nm and the 1500 nm GO. Adsorption predicts delivery enhancement independent of size and sedimentation. Error …
Figures 9A-C. Delivery by GO is A) dose-dependent, B) observable by cell coloration, and C) abolished without sufficient adsorption time. Co-incubation enabled the independent control of molecular and GO concentrations. The use of Magnevist in the mM 34724791 range is 100 -fold greater compared to …
Figures 10 A-B. Additional adsorption time studies.
Figure 9C showed that both molecular adsorption and delivery enhancement were abolished when a Magnevist- 150 nm GO mixture was added directly to media without allowing for adsorption time. The same result was found with a mixture of A) 9 + 1500 nm GO and B) Magnevist + 1500 nm GO. Experiments were …
Figure 11. 24 hour cytotoxicity assay using HeLa cells at increasing Magnevist and GO concentrations. No significant cytotoxicity was observed up to a Magnevist concentration of 35 mM and a GO concentration of 250 g/mL. The maximum GO dose required for cellular delivery enhancement is 20 g/mL, or an …
Figures 12A-B. C ytotoxicity of Magnevist. A) Viability of HeLa cells at increasing Magnevist concentration as measured by the Guava ViaCount Assay. Analysis includes floating cells at the end of the 24-hr incubation period. The fitted I C₅₀ was 80 mM. B) Same as in a) except analysis was performed …
Figure 13. The generalizability of GO co-incubation as a strategy to enhance cellular delivery was demonstrated in the KB cell line. Similar levels of delivery enhancement were achieved in KB compared to HeLa. This result generalized across 9 and(Magnevist 11) for both the 150 nm and the 1500 nm …
Figures 14A-B. Inherent variability in cell delivery by GO co-incubation. A) Magnevist cell labeling with and(without GO 150 nm) resulted in % CVs of 62 % over 17 trials and 28 % over 10 trials, respectively. B) A % CV of 23 % was measured across 3 batches of 100 -150 nm GO in the delivery of 9. …
Figures 15A-D. Sensitivity of the GO co-incubation protocol to procedural parameters. A) Cellular uptake of Magnevist with and without GO co-incubation decreases with cell density. B) Cellular Gd content decreases when incubating in a 6-well plate compared to a 24-well plate. C) 30 % 40 % of the …
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
Figure 17. Adsorption as characterized by measuring the amount of Gd left in the supe rn atant after the GO has been centrifuged into a pellet. These numbers are 30-1000 times greater than those measured directly from the pellet after washing. Direct measurement of adsorption on the GO pellet is …
Figure 5). From these results, the relative importance of different chemical groups in GO surface interaction and the factors controlling GO delivery performance were assessed. Table 1. GO Size Summary Square root of area (nm) Height (nm) Sample Mean Median Mean Median 150 nm GO 179.3 144.3 0.86 …
Figure 6). Therefore, forces outside of the amine-GO interaction contribute to GO adsorption, but their intricacies in complex media are presently beyond prediction by simple chemical variables. [00034] GO Sedimentation. Surface-adsorbing molecules have the potential to modify the sedimentation …
Figure 7B). 34724791 Table 3. Delivery of Gd(III)-labeled Molecules With and Without GO Mil ecu l e Gd ma ntent (fm$Al/eU utir 1 0 36 2 0. SVG 15251681.08-30-2016.ISHWT₉MFRXEAPX4.SPEC.10.3.727.692.740.709.svg 0.057 0.043 Chemistry Black and white 7 0. 36 0,63 SVG …
Figures 7C, 8, Table 4). The 34724791 regression coefficients for adsorption (b i), sedimentation (b 2), and size (b3) are 1. 0x10 2, -1.4 x10 3, and-7 -2X₁₀ 3, respectively, when the variables are expressed in units of Gd/10 5 C, AUC, and nm. The negative sign of b 3 indicates that cells …
Figure 9B). Second, direct addition of Magnevist and GO into media without a pre-incubation period in water eliminated both adsorption and delivery enhancement (
Figures 14 -15). The(labeling achieved using Magnevist alone 35 mM) and(with GO co-incubation 10 p g/mL) was 30 and 47 fmol Gd/cell, respectively. As expected, the labeling by Magnevist was comparable to the highest reported literature value, while that attained with GO represented a further …
Claims define the patent's legal scope. Independent claims stand alone; dependent claims (nested) narrow them. Click a claim to expand its dependents.
A method of bioactive molecule delivery, comprising: incubating cells with a co-incubation medium for 24 hours to deliver a bioactive molecular component into the cells, wherein said co-incubation medium comprises functionalized graphene oxide having said bioactive molecular component coupled on a surface of g raphene oxide (GO), wherein said bioactive molecular component i- s consists of Gd(III)-labeled molecules; and enhancing delivery of said bioactive molecular component into the cells by GO sedimentation. Currently amended
The method of claim 1, wherein said Gd(III)-labeled molecules comprise a moiety selected from alkyl, amine, hydroxy, phosphate, carboxy, quaternary amine and aminoalkyl moieties, and combinations thereof. Currently amended
The method of claim 1, wherein said bioactive molecular component is conjugated with an MR I contrast agent to monitor/quantify said delivery. Previously presented
The method of claim 1, wherein said graphene oxide has a lateral surface dimension of less than 1500 nm. Currently amended
The method of claim 1, wherein said cells comprise HeLa cells and/or KB cells. Previously presented
The method of claim 1, wherein said co-incubation medium has a cargo-to-GO ratio of 2. 1 x 10 6 Gd/10 5 C. Previously presented
,. Canceled
,,. Canceled
(Canceled,. Canceled
9-20.. Canceled
Canceled
,. Canceled
The method of claim [[1]] 27, wherein the period of time is 24 hours. Currently amended
,. Canceled
. Canceled
A method of bioactive molecule delivery, medium for a period of time to deliver a bioactive said co-incubation medium comprises functionalized molecular component coupled on a surface of molecular component comprises Gd(III)-labeled and GO in said co-incubation medium are 188 p comprising: incubating cells with a co-incubation molecular component into the cells, wherein graphene oxide having said bioactive graphene oxide (GO), wherein said bioactive molecules, and wherein concentrations of Gd(III) M and 18.8 p g/mL, respectively. New
Embodiments described in the patent, grouped by the materials and process steps they use.
3 materials1 process step
Synthesis of Gd(III)-labeled molecules (compounds 1-14) using copper(I)-catalyzed azide-alkyne click chemistry between Gd(HPN₃DO₃A) and alkyne-functionalized pendant groups, along with related synthetic routes. Purification and characterization performed by reverse-phase HPLC/MS and ESI-MS.
Materials described outside the worked examples.
graphene oxide
MRI contrast agent
Measurements and analyses referenced in the patent, with their drawing references.
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Performance values and ranges asserted in the specification or claims.
| Property | Value | Material |
|---|---|---|
Duration | 20.6–21.8 minutes | — |
Duration | 16.4–16.8 minutes |
Related documents with shared materials, methods, properties, or citations.
Patent
Atlas literature
Patent
US 10,994,016Patent drawings and their descriptions. Click a drawing to enlarge it.
Figure 1. The cellular delivery of bioactive molecules can be enhanced by simple mixing and co-incubation with graphene oxide in culture. [0
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 4. Structures of the 14 molecules tested with GO for adsorption, sedimentation, and cellular delivery. The quenching properties of GO usually hinder quantitative studies. The challenge is overcome by using Gd(III) to enable analytical quantitation by ICP. 34724791 [0
Figures 5A-C. A) GO adsorbed a(variety of molecules in cell culture media with amines 8, 9, 10, 12) producing the strongest interaction and(hydroxyls 2) and(phosphates 3) placing a far second. B) GO sedimentation induced by molecular adsorption was measured by the fraction of GO remaining in the …
Figures 6A-C. Adsorption as a function of A) charge, B) hydrophobicity, and C) hydrogen bonding potential of the adsorbing molecule on GO. No significant correlation was observed between adsorption and any of the chemical variables, indicating that GO adsorption in media cannot be simply predicted …
Figures 7A-C. Molecular adsorption, GO sedimentation, and GO size control the cellular delivery process. Molecules that A) exhibited higher adsorption and B) induced increased sedimentation experienced greater delivery enhancement by GO. Simple linear regression required separate fits for the two GO …
Figure 8. A significant correlation between delivery enhancement and adsorption can be found for molecules that did not increase GO sedimentation. The correlation was found for both the 150 nm and the 1500 nm GO. Adsorption predicts delivery enhancement independent of size and sedimentation. Error …
Figures 9A-C. Delivery by GO is A) dose-dependent, B) observable by cell coloration, and C) abolished without sufficient adsorption time. Co-incubation enabled the independent control of molecular and GO concentrations. The use of Magnevist in the mM 34724791 range is 100 -fold greater compared to …
Figures 10 A-B. Additional adsorption time studies.
Figure 9C showed that both molecular adsorption and delivery enhancement were abolished when a Magnevist- 150 nm GO mixture was added directly to media without allowing for adsorption time. The same result was found with a mixture of A) 9 + 1500 nm GO and B) Magnevist + 1500 nm GO. Experiments were …
Figure 11. 24 hour cytotoxicity assay using HeLa cells at increasing Magnevist and GO concentrations. No significant cytotoxicity was observed up to a Magnevist concentration of 35 mM and a GO concentration of 250 g/mL. The maximum GO dose required for cellular delivery enhancement is 20 g/mL, or an …
Figures 12A-B. C ytotoxicity of Magnevist. A) Viability of HeLa cells at increasing Magnevist concentration as measured by the Guava ViaCount Assay. Analysis includes floating cells at the end of the 24-hr incubation period. The fitted I C₅₀ was 80 mM. B) Same as in a) except analysis was performed …
Figure 13. The generalizability of GO co-incubation as a strategy to enhance cellular delivery was demonstrated in the KB cell line. Similar levels of delivery enhancement were achieved in KB compared to HeLa. This result generalized across 9 and(Magnevist 11) for both the 150 nm and the 1500 nm …
Figures 14A-B. Inherent variability in cell delivery by GO co-incubation. A) Magnevist cell labeling with and(without GO 150 nm) resulted in % CVs of 62 % over 17 trials and 28 % over 10 trials, respectively. B) A % CV of 23 % was measured across 3 batches of 100 -150 nm GO in the delivery of 9. …
Figures 15A-D. Sensitivity of the GO co-incubation protocol to procedural parameters. A) Cellular uptake of Magnevist with and without GO co-incubation decreases with cell density. B) Cellular Gd content decreases when incubating in a 6-well plate compared to a 24-well plate. C) 30 % 40 % of the …
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
Figure 17. Adsorption as characterized by measuring the amount of Gd left in the supe rn atant after the GO has been centrifuged into a pellet. These numbers are 30-1000 times greater than those measured directly from the pellet after washing. Direct measurement of adsorption on the GO pellet is …
Figure 5). From these results, the relative importance of different chemical groups in GO surface interaction and the factors controlling GO delivery performance were assessed. Table 1. GO Size Summary Square root of area (nm) Height (nm) Sample Mean Median Mean Median 150 nm GO 179.3 144.3 0.86 …
Figure 6). Therefore, forces outside of the amine-GO interaction contribute to GO adsorption, but their intricacies in complex media are presently beyond prediction by simple chemical variables. [00034] GO Sedimentation. Surface-adsorbing molecules have the potential to modify the sedimentation …
Figure 7B). 34724791 Table 3. Delivery of Gd(III)-labeled Molecules With and Without GO Mil ecu l e Gd ma ntent (fm$Al/eU utir 1 0 36 2 0. SVG 15251681.08-30-2016.ISHWT₉MFRXEAPX4.SPEC.10.3.727.692.740.709.svg 0.057 0.043 Chemistry Black and white 7 0. 36 0,63 SVG …
Figures 7C, 8, Table 4). The 34724791 regression coefficients for adsorption (b i), sedimentation (b 2), and size (b3) are 1. 0x10 2, -1.4 x10 3, and-7 -2X₁₀ 3, respectively, when the variables are expressed in units of Gd/10 5 C, AUC, and nm. The negative sign of b 3 indicates that cells …
Figure 9B). Second, direct addition of Magnevist and GO into media without a pre-incubation period in water eliminated both adsorption and delivery enhancement (
Figures 14 -15). The(labeling achieved using Magnevist alone 35 mM) and(with GO co-incubation 10 p g/mL) was 30 and 47 fmol Gd/cell, respectively. As expected, the labeling by Magnevist was comparable to the highest reported literature value, while that attained with GO represented a further …
Claims define the patent's legal scope. Independent claims stand alone; dependent claims (nested) narrow them. Click a claim to expand its dependents.
A method of bioactive molecule delivery, comprising: incubating cells with a co-incubation medium for 24 hours to deliver a bioactive molecular component into the cells, wherein said co-incubation medium comprises functionalized graphene oxide having said bioactive molecular component coupled on a surface of g raphene oxide (GO), wherein said bioactive molecular component i- s consists of Gd(III)-labeled molecules; and enhancing delivery of said bioactive molecular component into the cells by GO sedimentation. Currently amended
The method of claim 1, wherein said Gd(III)-labeled molecules comprise a moiety selected from alkyl, amine, hydroxy, phosphate, carboxy, quaternary amine and aminoalkyl moieties, and combinations thereof. Currently amended
The method of claim 1, wherein said bioactive molecular component is conjugated with an MR I contrast agent to monitor/quantify said delivery. Previously presented
The method of claim 1, wherein said graphene oxide has a lateral surface dimension of less than 1500 nm. Currently amended
The method of claim 1, wherein said cells comprise HeLa cells and/or KB cells. Previously presented
The method of claim 1, wherein said co-incubation medium has a cargo-to-GO ratio of 2. 1 x 10 6 Gd/10 5 C. Previously presented
,. Canceled
,,. Canceled
(Canceled,. Canceled
9-20.. Canceled
Canceled
,. Canceled
The method of claim [[1]] 27, wherein the period of time is 24 hours. Currently amended
,. Canceled
. Canceled
A method of bioactive molecule delivery, medium for a period of time to deliver a bioactive said co-incubation medium comprises functionalized molecular component coupled on a surface of molecular component comprises Gd(III)-labeled and GO in said co-incubation medium are 188 p comprising: incubating cells with a co-incubation molecular component into the cells, wherein graphene oxide having said bioactive graphene oxide (GO), wherein said bioactive molecules, and wherein concentrations of Gd(III) M and 18.8 p g/mL, respectively. New
Embodiments described in the patent, grouped by the materials and process steps they use.
3 materials1 process step
Synthesis of Gd(III)-labeled molecules (compounds 1-14) using copper(I)-catalyzed azide-alkyne click chemistry between Gd(HPN₃DO₃A) and alkyne-functionalized pendant groups, along with related synthetic routes. Purification and characterization performed by reverse-phase HPLC/MS and ESI-MS.
Materials described outside the worked examples.
graphene oxide
MRI contrast agent
Measurements and analyses referenced in the patent, with their drawing references.
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Performance values and ranges asserted in the specification or claims.
| Property | Value | Material |
|---|---|---|
Duration | 20.6–21.8 minutes | — |
Duration | 16.4–16.8 minutes |
Related documents with shared materials, methods, properties, or citations.
Patent
Atlas literature
Patent
US 10,994,016Patent drawings and their descriptions. Click a drawing to enlarge it.
Figure 1. The cellular delivery of bioactive molecules can be enhanced by simple mixing and co-incubation with graphene oxide in culture. [0
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 4. Structures of the 14 molecules tested with GO for adsorption, sedimentation, and cellular delivery. The quenching properties of GO usually hinder quantitative studies. The challenge is overcome by using Gd(III) to enable analytical quantitation by ICP. 34724791 [0
Figures 5A-C. A) GO adsorbed a(variety of molecules in cell culture media with amines 8, 9, 10, 12) producing the strongest interaction and(hydroxyls 2) and(phosphates 3) placing a far second. B) GO sedimentation induced by molecular adsorption was measured by the fraction of GO remaining in the …
Figures 6A-C. Adsorption as a function of A) charge, B) hydrophobicity, and C) hydrogen bonding potential of the adsorbing molecule on GO. No significant correlation was observed between adsorption and any of the chemical variables, indicating that GO adsorption in media cannot be simply predicted …
Figures 7A-C. Molecular adsorption, GO sedimentation, and GO size control the cellular delivery process. Molecules that A) exhibited higher adsorption and B) induced increased sedimentation experienced greater delivery enhancement by GO. Simple linear regression required separate fits for the two GO …
Figure 8. A significant correlation between delivery enhancement and adsorption can be found for molecules that did not increase GO sedimentation. The correlation was found for both the 150 nm and the 1500 nm GO. Adsorption predicts delivery enhancement independent of size and sedimentation. Error …
Figures 9A-C. Delivery by GO is A) dose-dependent, B) observable by cell coloration, and C) abolished without sufficient adsorption time. Co-incubation enabled the independent control of molecular and GO concentrations. The use of Magnevist in the mM 34724791 range is 100 -fold greater compared to …
Figures 10 A-B. Additional adsorption time studies.
Figure 9C showed that both molecular adsorption and delivery enhancement were abolished when a Magnevist- 150 nm GO mixture was added directly to media without allowing for adsorption time. The same result was found with a mixture of A) 9 + 1500 nm GO and B) Magnevist + 1500 nm GO. Experiments were …
Figure 11. 24 hour cytotoxicity assay using HeLa cells at increasing Magnevist and GO concentrations. No significant cytotoxicity was observed up to a Magnevist concentration of 35 mM and a GO concentration of 250 g/mL. The maximum GO dose required for cellular delivery enhancement is 20 g/mL, or an …
Figures 12A-B. C ytotoxicity of Magnevist. A) Viability of HeLa cells at increasing Magnevist concentration as measured by the Guava ViaCount Assay. Analysis includes floating cells at the end of the 24-hr incubation period. The fitted I C₅₀ was 80 mM. B) Same as in a) except analysis was performed …
Figure 13. The generalizability of GO co-incubation as a strategy to enhance cellular delivery was demonstrated in the KB cell line. Similar levels of delivery enhancement were achieved in KB compared to HeLa. This result generalized across 9 and(Magnevist 11) for both the 150 nm and the 1500 nm …
Figures 14A-B. Inherent variability in cell delivery by GO co-incubation. A) Magnevist cell labeling with and(without GO 150 nm) resulted in % CVs of 62 % over 17 trials and 28 % over 10 trials, respectively. B) A % CV of 23 % was measured across 3 batches of 100 -150 nm GO in the delivery of 9. …
Figures 15A-D. Sensitivity of the GO co-incubation protocol to procedural parameters. A) Cellular uptake of Magnevist with and without GO co-incubation decreases with cell density. B) Cellular Gd content decreases when incubating in a 6-well plate compared to a 24-well plate. C) 30 % 40 % of the …
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
Figure 17. Adsorption as characterized by measuring the amount of Gd left in the supe rn atant after the GO has been centrifuged into a pellet. These numbers are 30-1000 times greater than those measured directly from the pellet after washing. Direct measurement of adsorption on the GO pellet is …
Figure 5). From these results, the relative importance of different chemical groups in GO surface interaction and the factors controlling GO delivery performance were assessed. Table 1. GO Size Summary Square root of area (nm) Height (nm) Sample Mean Median Mean Median 150 nm GO 179.3 144.3 0.86 …
Figure 6). Therefore, forces outside of the amine-GO interaction contribute to GO adsorption, but their intricacies in complex media are presently beyond prediction by simple chemical variables. [00034] GO Sedimentation. Surface-adsorbing molecules have the potential to modify the sedimentation …
Figure 7B). 34724791 Table 3. Delivery of Gd(III)-labeled Molecules With and Without GO Mil ecu l e Gd ma ntent (fm$Al/eU utir 1 0 36 2 0. SVG 15251681.08-30-2016.ISHWT₉MFRXEAPX4.SPEC.10.3.727.692.740.709.svg 0.057 0.043 Chemistry Black and white 7 0. 36 0,63 SVG …
Figures 7C, 8, Table 4). The 34724791 regression coefficients for adsorption (b i), sedimentation (b 2), and size (b3) are 1. 0x10 2, -1.4 x10 3, and-7 -2X₁₀ 3, respectively, when the variables are expressed in units of Gd/10 5 C, AUC, and nm. The negative sign of b 3 indicates that cells …
Figure 9B). Second, direct addition of Magnevist and GO into media without a pre-incubation period in water eliminated both adsorption and delivery enhancement (
Figures 14 -15). The(labeling achieved using Magnevist alone 35 mM) and(with GO co-incubation 10 p g/mL) was 30 and 47 fmol Gd/cell, respectively. As expected, the labeling by Magnevist was comparable to the highest reported literature value, while that attained with GO represented a further …
Claims define the patent's legal scope. Independent claims stand alone; dependent claims (nested) narrow them. Click a claim to expand its dependents.
A method of bioactive molecule delivery, comprising: incubating cells with a co-incubation medium for 24 hours to deliver a bioactive molecular component into the cells, wherein said co-incubation medium comprises functionalized graphene oxide having said bioactive molecular component coupled on a surface of g raphene oxide (GO), wherein said bioactive molecular component i- s consists of Gd(III)-labeled molecules; and enhancing delivery of said bioactive molecular component into the cells by GO sedimentation. Currently amended
The method of claim 1, wherein said Gd(III)-labeled molecules comprise a moiety selected from alkyl, amine, hydroxy, phosphate, carboxy, quaternary amine and aminoalkyl moieties, and combinations thereof. Currently amended
The method of claim 1, wherein said bioactive molecular component is conjugated with an MR I contrast agent to monitor/quantify said delivery. Previously presented
The method of claim 1, wherein said graphene oxide has a lateral surface dimension of less than 1500 nm. Currently amended
The method of claim 1, wherein said cells comprise HeLa cells and/or KB cells. Previously presented
The method of claim 1, wherein said co-incubation medium has a cargo-to-GO ratio of 2. 1 x 10 6 Gd/10 5 C. Previously presented
,. Canceled
,,. Canceled
(Canceled,. Canceled
9-20.. Canceled
Canceled
,. Canceled
The method of claim [[1]] 27, wherein the period of time is 24 hours. Currently amended
,. Canceled
. Canceled
A method of bioactive molecule delivery, medium for a period of time to deliver a bioactive said co-incubation medium comprises functionalized molecular component coupled on a surface of molecular component comprises Gd(III)-labeled and GO in said co-incubation medium are 188 p comprising: incubating cells with a co-incubation molecular component into the cells, wherein graphene oxide having said bioactive graphene oxide (GO), wherein said bioactive molecules, and wherein concentrations of Gd(III) M and 18.8 p g/mL, respectively. New
Embodiments described in the patent, grouped by the materials and process steps they use.
3 materials1 process step
Synthesis of Gd(III)-labeled molecules (compounds 1-14) using copper(I)-catalyzed azide-alkyne click chemistry between Gd(HPN₃DO₃A) and alkyne-functionalized pendant groups, along with related synthetic routes. Purification and characterization performed by reverse-phase HPLC/MS and ESI-MS.
Materials described outside the worked examples.
graphene oxide
MRI contrast agent
Measurements and analyses referenced in the patent, with their drawing references.
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Performance values and ranges asserted in the specification or claims.
| Property | Value | Material |
|---|---|---|
Duration | 20.6–21.8 minutes | — |
Duration | 16.4–16.8 minutes |
Related documents with shared materials, methods, properties, or citations.
Patent
Atlas literature
Patent
US 10,994,016Patent drawings and their descriptions. Click a drawing to enlarge it.
Figure 1. The cellular delivery of bioactive molecules can be enhanced by simple mixing and co-incubation with graphene oxide in culture. [0
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 4. Structures of the 14 molecules tested with GO for adsorption, sedimentation, and cellular delivery. The quenching properties of GO usually hinder quantitative studies. The challenge is overcome by using Gd(III) to enable analytical quantitation by ICP. 34724791 [0
Figures 5A-C. A) GO adsorbed a(variety of molecules in cell culture media with amines 8, 9, 10, 12) producing the strongest interaction and(hydroxyls 2) and(phosphates 3) placing a far second. B) GO sedimentation induced by molecular adsorption was measured by the fraction of GO remaining in the …
Figures 6A-C. Adsorption as a function of A) charge, B) hydrophobicity, and C) hydrogen bonding potential of the adsorbing molecule on GO. No significant correlation was observed between adsorption and any of the chemical variables, indicating that GO adsorption in media cannot be simply predicted …
Figures 7A-C. Molecular adsorption, GO sedimentation, and GO size control the cellular delivery process. Molecules that A) exhibited higher adsorption and B) induced increased sedimentation experienced greater delivery enhancement by GO. Simple linear regression required separate fits for the two GO …
Figure 8. A significant correlation between delivery enhancement and adsorption can be found for molecules that did not increase GO sedimentation. The correlation was found for both the 150 nm and the 1500 nm GO. Adsorption predicts delivery enhancement independent of size and sedimentation. Error …
Figures 9A-C. Delivery by GO is A) dose-dependent, B) observable by cell coloration, and C) abolished without sufficient adsorption time. Co-incubation enabled the independent control of molecular and GO concentrations. The use of Magnevist in the mM 34724791 range is 100 -fold greater compared to …
Figures 10 A-B. Additional adsorption time studies.
Figure 9C showed that both molecular adsorption and delivery enhancement were abolished when a Magnevist- 150 nm GO mixture was added directly to media without allowing for adsorption time. The same result was found with a mixture of A) 9 + 1500 nm GO and B) Magnevist + 1500 nm GO. Experiments were …
Figure 11. 24 hour cytotoxicity assay using HeLa cells at increasing Magnevist and GO concentrations. No significant cytotoxicity was observed up to a Magnevist concentration of 35 mM and a GO concentration of 250 g/mL. The maximum GO dose required for cellular delivery enhancement is 20 g/mL, or an …
Figures 12A-B. C ytotoxicity of Magnevist. A) Viability of HeLa cells at increasing Magnevist concentration as measured by the Guava ViaCount Assay. Analysis includes floating cells at the end of the 24-hr incubation period. The fitted I C₅₀ was 80 mM. B) Same as in a) except analysis was performed …
Figure 13. The generalizability of GO co-incubation as a strategy to enhance cellular delivery was demonstrated in the KB cell line. Similar levels of delivery enhancement were achieved in KB compared to HeLa. This result generalized across 9 and(Magnevist 11) for both the 150 nm and the 1500 nm …
Figures 14A-B. Inherent variability in cell delivery by GO co-incubation. A) Magnevist cell labeling with and(without GO 150 nm) resulted in % CVs of 62 % over 17 trials and 28 % over 10 trials, respectively. B) A % CV of 23 % was measured across 3 batches of 100 -150 nm GO in the delivery of 9. …
Figures 15A-D. Sensitivity of the GO co-incubation protocol to procedural parameters. A) Cellular uptake of Magnevist with and without GO co-incubation decreases with cell density. B) Cellular Gd content decreases when incubating in a 6-well plate compared to a 24-well plate. C) 30 % 40 % of the …
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
Figure 17. Adsorption as characterized by measuring the amount of Gd left in the supe rn atant after the GO has been centrifuged into a pellet. These numbers are 30-1000 times greater than those measured directly from the pellet after washing. Direct measurement of adsorption on the GO pellet is …
Figure 5). From these results, the relative importance of different chemical groups in GO surface interaction and the factors controlling GO delivery performance were assessed. Table 1. GO Size Summary Square root of area (nm) Height (nm) Sample Mean Median Mean Median 150 nm GO 179.3 144.3 0.86 …
Figure 6). Therefore, forces outside of the amine-GO interaction contribute to GO adsorption, but their intricacies in complex media are presently beyond prediction by simple chemical variables. [00034] GO Sedimentation. Surface-adsorbing molecules have the potential to modify the sedimentation …
Figure 7B). 34724791 Table 3. Delivery of Gd(III)-labeled Molecules With and Without GO Mil ecu l e Gd ma ntent (fm$Al/eU utir 1 0 36 2 0. SVG 15251681.08-30-2016.ISHWT₉MFRXEAPX4.SPEC.10.3.727.692.740.709.svg 0.057 0.043 Chemistry Black and white 7 0. 36 0,63 SVG …
Figures 7C, 8, Table 4). The 34724791 regression coefficients for adsorption (b i), sedimentation (b 2), and size (b3) are 1. 0x10 2, -1.4 x10 3, and-7 -2X₁₀ 3, respectively, when the variables are expressed in units of Gd/10 5 C, AUC, and nm. The negative sign of b 3 indicates that cells …
Figure 9B). Second, direct addition of Magnevist and GO into media without a pre-incubation period in water eliminated both adsorption and delivery enhancement (
Figures 14 -15). The(labeling achieved using Magnevist alone 35 mM) and(with GO co-incubation 10 p g/mL) was 30 and 47 fmol Gd/cell, respectively. As expected, the labeling by Magnevist was comparable to the highest reported literature value, while that attained with GO represented a further …
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A method of bioactive molecule delivery, comprising: incubating cells with a co-incubation medium for 24 hours to deliver a bioactive molecular component into the cells, wherein said co-incubation medium comprises functionalized graphene oxide having said bioactive molecular component coupled on a surface of g raphene oxide (GO), wherein said bioactive molecular component i- s consists of Gd(III)-labeled molecules; and enhancing delivery of said bioactive molecular component into the cells by GO sedimentation. Currently amended
The method of claim 1, wherein said Gd(III)-labeled molecules comprise a moiety selected from alkyl, amine, hydroxy, phosphate, carboxy, quaternary amine and aminoalkyl moieties, and combinations thereof. Currently amended
The method of claim 1, wherein said bioactive molecular component is conjugated with an MR I contrast agent to monitor/quantify said delivery. Previously presented
The method of claim 1, wherein said graphene oxide has a lateral surface dimension of less than 1500 nm. Currently amended
The method of claim 1, wherein said cells comprise HeLa cells and/or KB cells. Previously presented
The method of claim 1, wherein said co-incubation medium has a cargo-to-GO ratio of 2. 1 x 10 6 Gd/10 5 C. Previously presented
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The method of claim [[1]] 27, wherein the period of time is 24 hours. Currently amended
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A method of bioactive molecule delivery, medium for a period of time to deliver a bioactive said co-incubation medium comprises functionalized molecular component coupled on a surface of molecular component comprises Gd(III)-labeled and GO in said co-incubation medium are 188 p comprising: incubating cells with a co-incubation molecular component into the cells, wherein graphene oxide having said bioactive graphene oxide (GO), wherein said bioactive molecules, and wherein concentrations of Gd(III) M and 18.8 p g/mL, respectively. New
Embodiments described in the patent, grouped by the materials and process steps they use.
3 materials1 process step
Synthesis of Gd(III)-labeled molecules (compounds 1-14) using copper(I)-catalyzed azide-alkyne click chemistry between Gd(HPN₃DO₃A) and alkyne-functionalized pendant groups, along with related synthetic routes. Purification and characterization performed by reverse-phase HPLC/MS and ESI-MS.
Materials described outside the worked examples.
graphene oxide
MRI contrast agent
Measurements and analyses referenced in the patent, with their drawing references.
Figure 2. Raman spectroscopy of the two graphene oxide preparations used for cellular delivery studies. Spectra are vertically staggered for clarity. The approximate Raman shift of the D, G, 2D and D+G bands are 1350 cm- 1, 1584 cm- 1, 2700 cm- 1, and 2934 cm- 1, respectively. [0
Performance values and ranges asserted in the specification or claims.
| Property | Value | Material |
|---|---|---|
Duration | 20.6–21.8 minutes | — |
Duration | 16.4–16.8 minutes |
Related documents with shared materials, methods, properties, or citations.
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
| — |
Duration | 10.4–12.5 minutes | — |
Duration | 10.2–11.3 minutes | — |
Duration | 4.7–6.3 minutes | — |
Duration | 25.4–26.4 minutes | — |
Duration | 15.9–16.6 minutes | — |
Thickness | 1100–3500 cm | — |
Duration | 30–60 minutes | — |
Temperature | ≤ 20 °C | — |
Duration | ≥ 15 minutes | — |
Duration | ≥ 5 minutes | — |
Thickness | ≤ 1500 nm | — |
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
| — |
Duration | 10.4–12.5 minutes | — |
Duration | 10.2–11.3 minutes | — |
Duration | 4.7–6.3 minutes | — |
Duration | 25.4–26.4 minutes | — |
Duration | 15.9–16.6 minutes | — |
Thickness | 1100–3500 cm | — |
Duration | 30–60 minutes | — |
Temperature | ≤ 20 °C | — |
Duration | ≥ 15 minutes | — |
Duration | ≥ 5 minutes | — |
Thickness | ≤ 1500 nm | — |
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
| — |
Duration | 10.4–12.5 minutes | — |
Duration | 10.2–11.3 minutes | — |
Duration | 4.7–6.3 minutes | — |
Duration | 25.4–26.4 minutes | — |
Duration | 15.9–16.6 minutes | — |
Thickness | 1100–3500 cm | — |
Duration | 30–60 minutes | — |
Temperature | ≤ 20 °C | — |
Duration | ≥ 15 minutes | — |
Duration | ≥ 5 minutes | — |
Thickness | ≤ 1500 nm | — |
Figure 3. X-ray photoelectron spectroscopy of the two graphene oxide preparations used for cellular delivery studies. The 150 nm GO contains 10 %-15% more oxygen groups compared to the 1500 nm GO. [0
Figure 16. GO co-incubation enhanced (digital images) the sensitivity of Magnevist-labeled cells from 5000 cells/pL to 500 cells/pL on T 1 -weighted MRI at 7T. For comparison, a packed cell pellet has approximately 385,000 cells/L using the reported HeLa cellular volume of 2.6 pL. The 150 nm GO was …
| — |
Duration | 10.4–12.5 minutes | — |
Duration | 10.2–11.3 minutes | — |
Duration | 4.7–6.3 minutes | — |
Duration | 25.4–26.4 minutes | — |
Duration | 15.9–16.6 minutes | — |
Thickness | 1100–3500 cm | — |
Duration | 30–60 minutes | — |
Temperature | ≤ 20 °C | — |
Duration | ≥ 15 minutes | — |
Duration | ≥ 5 minutes | — |
Thickness | ≤ 1500 nm | — |