Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution

Haley R. Phillips; Zachary P. Tolstyka; Bryan C. Hall; Joseph K. Hexum; Perry B Hackett; Theresa M Reineke

doi:10.1021/acs.biomac.8b01704

Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution

Haley R. Phillips, Zachary P. Tolstyka, Bryan C. Hall, Joseph K. Hexum, Perry B Hackett, Theresa M Reineke

Research output: Contribution to journal › Article › peer-review

14 Scopus citations

Abstract

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) [P(MAG-b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Original language	English (US)
Pages (from-to)	1530-1544
Number of pages	15
Journal	Biomacromolecules
Volume	20
Issue number	4
DOIs	https://doi.org/10.1021/acs.biomac.8b01704
State	Published - Apr 8 2019

Bibliographical note

Funding Information:
The authors acknowledge the NIH Director’s New Innovator Program (DP2OD006669) and the Camille and Henry Dreyfus Foundation for funding this work. UMN Bruker NMR spectrometer research reported in this publication was supported by the Office of the Director, National Institutes of Health of the National Institutes of Health under Award Number S10OD011952. We also acknowledge the financial support from NIH grants 1R01DK082516 and P01HD32652. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors also thank the Fairview Hospital Special Coagulation Clinic and the University Genomics Core at the University of Minnesota for research support. We also acknowledge work done using the IVIS Spectrum in vivo imaging system at the University of Minnesota−University Imaging Centers, http://uic.umn.edu.

Publisher Copyright:
© 2019 American Chemical Society.

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title = "Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution",

abstract = "Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) [P(MAG-b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.",

author = "Phillips, {Haley R.} and Tolstyka, {Zachary P.} and Hall, {Bryan C.} and Hexum, {Joseph K.} and Hackett, {Perry B} and Reineke, {Theresa M}",

note = "Funding Information: The authors acknowledge the NIH Director{\textquoteright}s New Innovator Program (DP2OD006669) and the Camille and Henry Dreyfus Foundation for funding this work. UMN Bruker NMR spectrometer research reported in this publication was supported by the Office of the Director, National Institutes of Health of the National Institutes of Health under Award Number S10OD011952. We also acknowledge the financial support from NIH grants 1R01DK082516 and P01HD32652. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors also thank the Fairview Hospital Special Coagulation Clinic and the University Genomics Core at the University of Minnesota for research support. We also acknowledge work done using the IVIS Spectrum in vivo imaging system at the University of Minnesota−University Imaging Centers, http://uic.umn.edu. Publisher Copyright: {\textcopyright} 2019 American Chemical Society.",

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AU - Hall, Bryan C.

AU - Hexum, Joseph K.

AU - Hackett, Perry B

AU - Reineke, Theresa M

N1 - Funding Information: The authors acknowledge the NIH Director’s New Innovator Program (DP2OD006669) and the Camille and Henry Dreyfus Foundation for funding this work. UMN Bruker NMR spectrometer research reported in this publication was supported by the Office of the Director, National Institutes of Health of the National Institutes of Health under Award Number S10OD011952. We also acknowledge the financial support from NIH grants 1R01DK082516 and P01HD32652. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors also thank the Fairview Hospital Special Coagulation Clinic and the University Genomics Core at the University of Minnesota for research support. We also acknowledge work done using the IVIS Spectrum in vivo imaging system at the University of Minnesota−University Imaging Centers, http://uic.umn.edu. Publisher Copyright: © 2019 American Chemical Society.

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N2 - Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) [P(MAG-b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

AB - Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) [P(MAG-b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

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Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution

Abstract

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IVIS Spectrum In Vivo Imaging System

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Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution

Abstract

Bibliographical note

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IVIS Spectrum In Vivo Imaging System

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