DNA
Supporting Information
Critical length of PEG grafts on lPEI/DNA nanoparticles for efficient in vivo delivery John-Michael Williford,†,‡ # Maani M. Archang,§,‡,# Il Minn,|| Yong Ren,§,‡ Mark Wo,§,‡ John Vandermark,§,‡ Paul B. Fisher,a,b,c Martin G. Pomper,||,§,‡ and Hai-Quan Mao§,‡,^,* †
Department of Biomedical Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA ‡
Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218,
USA §
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore,
MD 21218, USA ||
Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins
Medical Institutions, Baltimore, MD 21287, USA ^
Translational Tissue Engineering Center and Whitaker Biomedical Engineering Institute,
Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA a
Department of Human and Molecular Genetics, Virginia Commonwealth University,
Richmond, VA 23298, USA b
VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond,
VA 23298, USA c
VCU Massey Cancer Center, Virginia Commonwealth University, Richmond, VA
23298, USA #
These authors contributed equally to this work.
*Correspondence should be addressed to [email protected].
S1
Figure S1: 1H NMR spectrum of lPEI-g-PEG5H with a grafting degree of 2.3%. Unreacted PEG5H was removed by ultrafiltration with 3,000 MW cutoff. Peaks a, b, c represent protons in the pyridine ring on the copolymers. Since lPEI was partially charged, the -CH2CH2- on the backbone had two peaks. One peak was at δ = 3.2 and the other overlapped with PEG at δ = 3.6.
S2
Figure S2: Effect of N/P ratio on (A) transfection efficiency and (B) metabolic activity of lPEI-g-PEG7H/DNA nanoparticles, lPEI-g-PEG2K/DNA nanoparticles, and lPEI control nanoparticles in PC3-ML cells. 0.2% PEG grafting degree was used for comparison. Each bar represents mean ± standard deviation (n = 3).
Figure S3: Average major and minor axis lengths (A) and aspect ratios (B) of lPEI-gPEG7H/DNA nanoparticles and lPEI-g-PEG2K/DNA nanoparticles prepared with different grafting degrees. Each bar represents mean ± standard deviation (n > 100 particles). S3
Figure S4: Transfection efficiency of lPEI-g-PEG7H/DNA and lPEI-g-PEG2K/DNA nanoparticles in MDA-MB-231 cells (A) and HeLa cells (B). Each bar represents mean ± standard deviation (n = 3). ** p < 0.01, *** p < 0.001.
S4
Figure S5: DNA release from lPEI-g-PEG7H/DNA and lPEI-g-PEG2K/DNA nanoparticles prepared with 0.2% PEG grafting degree (A), 0.5% PEG grafting degree (B), 1% PEG grafting degree (C), and 2.3% PEG grafting degree (D) after treatment with increasing concentrations of heparin sulfate in 150 mM NaCl solution for 15 min. Each point represents mean ± standard deviation (n = 3).
S5
Figure S6: Comparison of nanoparticle size (A) and in vitro transfection efficiency (B) of lPEI-g-PEG7H/DNA nanoparticles and lPEI-g-PEG5H/DNA nanoparticles at 0.2% and 1% PEG grafting degrees. Each bar represents mean ± standard deviation (n = 3).
S6
Figure S7: Transfection efficiency of lPEI-g-PEG5H/DNA (A) and lPEI-g-PEG2K/DNA (B) nanoparticles in MDA-MB-231-αvβ3+ cells comparing nanoparticles with and without conjugation of cell binding peptide cRGD. Each bar represents mean ± standard deviation (n = 3). * p < 0.05.
S7
Figure S8: H&E staining of tumor sections demonstrating the presence of PC3-ML metastatic lesions in the lung, liver, and kidney for all nanoparticle treatment groups. Tumor regions are marked with the T.
S8
Critical length of PEG grafts on lPEI/DNA nanoparticles for efficient in vivo delivery John-Michael Williford,†,‡ # Maani M. Archang,§,‡,# Il Minn,|| Yong Ren,§,‡ Mark Wo,§,‡ John Vandermark,§,‡ Paul B. Fisher,a,b,c Martin G. Pomper,||,§,‡ and Hai-Quan Mao§,‡,^,* †
Department of Biomedical Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA ‡
Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218,
USA §
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore,
MD 21218, USA ||
Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins
Medical Institutions, Baltimore, MD 21287, USA ^
Translational Tissue Engineering Center and Whitaker Biomedical Engineering Institute,
Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA a
Department of Human and Molecular Genetics, Virginia Commonwealth University,
Richmond, VA 23298, USA b
VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond,
VA 23298, USA c
VCU Massey Cancer Center, Virginia Commonwealth University, Richmond, VA
23298, USA #
These authors contributed equally to this work.
*Correspondence should be addressed to [email protected].
S1
Figure S1: 1H NMR spectrum of lPEI-g-PEG5H with a grafting degree of 2.3%. Unreacted PEG5H was removed by ultrafiltration with 3,000 MW cutoff. Peaks a, b, c represent protons in the pyridine ring on the copolymers. Since lPEI was partially charged, the -CH2CH2- on the backbone had two peaks. One peak was at δ = 3.2 and the other overlapped with PEG at δ = 3.6.
S2
Figure S2: Effect of N/P ratio on (A) transfection efficiency and (B) metabolic activity of lPEI-g-PEG7H/DNA nanoparticles, lPEI-g-PEG2K/DNA nanoparticles, and lPEI control nanoparticles in PC3-ML cells. 0.2% PEG grafting degree was used for comparison. Each bar represents mean ± standard deviation (n = 3).
Figure S3: Average major and minor axis lengths (A) and aspect ratios (B) of lPEI-gPEG7H/DNA nanoparticles and lPEI-g-PEG2K/DNA nanoparticles prepared with different grafting degrees. Each bar represents mean ± standard deviation (n > 100 particles). S3
Figure S4: Transfection efficiency of lPEI-g-PEG7H/DNA and lPEI-g-PEG2K/DNA nanoparticles in MDA-MB-231 cells (A) and HeLa cells (B). Each bar represents mean ± standard deviation (n = 3). ** p < 0.01, *** p < 0.001.
S4
Figure S5: DNA release from lPEI-g-PEG7H/DNA and lPEI-g-PEG2K/DNA nanoparticles prepared with 0.2% PEG grafting degree (A), 0.5% PEG grafting degree (B), 1% PEG grafting degree (C), and 2.3% PEG grafting degree (D) after treatment with increasing concentrations of heparin sulfate in 150 mM NaCl solution for 15 min. Each point represents mean ± standard deviation (n = 3).
S5
Figure S6: Comparison of nanoparticle size (A) and in vitro transfection efficiency (B) of lPEI-g-PEG7H/DNA nanoparticles and lPEI-g-PEG5H/DNA nanoparticles at 0.2% and 1% PEG grafting degrees. Each bar represents mean ± standard deviation (n = 3).
S6
Figure S7: Transfection efficiency of lPEI-g-PEG5H/DNA (A) and lPEI-g-PEG2K/DNA (B) nanoparticles in MDA-MB-231-αvβ3+ cells comparing nanoparticles with and without conjugation of cell binding peptide cRGD. Each bar represents mean ± standard deviation (n = 3). * p < 0.05.
S7
Figure S8: H&E staining of tumor sections demonstrating the presence of PC3-ML metastatic lesions in the lung, liver, and kidney for all nanoparticle treatment groups. Tumor regions are marked with the T.
S8
