Novel water insoluble lipoparticulates for gene delivery

D. Y. Furgeson, R. N. Cohen, Ram I Mahato, S. W. Kim

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Purpose. The objective was to design and prepare water insoluble lipoparticulates (ISLPs) for efficient gene delivery to lung tissue. Methods. Nona{(ethylenimine)-co-[(2-aminoethyl)-N-choleseteryloxycarbonyl- ethylenimine]} (NEACE-T) was synthesized in both its free-base and chloride salt-forms using linear polyethylenimine (PEI, Mw 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitelechelic Nα-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by 1H-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a secondary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a colipid, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1/1, 1/2, and 2/1 molar ratios and the lipoparticulates were hydrated and filtered. ISLP/p2CMVmIL-12 complexes were characterized for particle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Results. Transgene expression was dependent on the site of cholesterol conjugation, lipononamer:colipid molar ratio, and ISLP/p2CMVmIL-12 charge ratios. ISLP/p2CMVmIL-12 complexes were nontoxic to murine colon adenocarcinoma (CT-26) cells at 9/1 (±) or lower, had a mean particle size of 330-400 nm while the ζ potential varied from 36-39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size comparable to that determined by dynamic light scattering. ISLP/p2CMVmIL-12 complexes prepared using free-base NEACE-T:DOPE (1/2) at charge ratios of 3/1 and 5/1 (±) provided the highest levels of transgene expression, 18 times more than the levels provided by the salt-form. Secreted levels of mIL-12 p70 were 75 times higher for ISLP/p2CMVmIL-12 complexes than naked p2CMVmIL-12 and nearly 4 times higher than PEI 25 kDa/p2CMVmIL-12 complexes. Conclusions. The transfection efficiency of the ISLPs was dependent on the site of cholesterol conjugation, amount of colipid, and charge ratio. The highest levels of transgene expression were provided by NEACE-T:DOPE (1/2)/p2CMVmIL-12 at a 3/1 (±) charge ratio.

Original languageEnglish (US)
Pages (from-to)382-390
Number of pages9
JournalPharmaceutical Research
Volume19
Issue number4
DOIs
StatePublished - Apr 29 2002

Fingerprint

Transgenes
Polyetherimides
Genes
Particle Size
Amines
Surface morphology
Transfection
Water
Salts
Particle size
Cholesterol
Polyethyleneimine
Atomic Force Microscopy
Dynamic light scattering
Zeta potential
Cytotoxicity
Anchors
Chlorides
Atomic force microscopy
Colon

Keywords

  • Cytokine
  • Insoluble
  • Lipononamer
  • Lipoparticulates
  • Transgene

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)

Cite this

Novel water insoluble lipoparticulates for gene delivery. / Furgeson, D. Y.; Cohen, R. N.; Mahato, Ram I; Kim, S. W.

In: Pharmaceutical Research, Vol. 19, No. 4, 29.04.2002, p. 382-390.

Research output: Contribution to journalArticle

Furgeson, D. Y. ; Cohen, R. N. ; Mahato, Ram I ; Kim, S. W. / Novel water insoluble lipoparticulates for gene delivery. In: Pharmaceutical Research. 2002 ; Vol. 19, No. 4. pp. 382-390.
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abstract = "Purpose. The objective was to design and prepare water insoluble lipoparticulates (ISLPs) for efficient gene delivery to lung tissue. Methods. Nona{(ethylenimine)-co-[(2-aminoethyl)-N-choleseteryloxycarbonyl- ethylenimine]} (NEACE-T) was synthesized in both its free-base and chloride salt-forms using linear polyethylenimine (PEI, Mw 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitelechelic Nα-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by 1H-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a secondary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a colipid, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1/1, 1/2, and 2/1 molar ratios and the lipoparticulates were hydrated and filtered. ISLP/p2CMVmIL-12 complexes were characterized for particle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Results. Transgene expression was dependent on the site of cholesterol conjugation, lipononamer:colipid molar ratio, and ISLP/p2CMVmIL-12 charge ratios. ISLP/p2CMVmIL-12 complexes were nontoxic to murine colon adenocarcinoma (CT-26) cells at 9/1 (±) or lower, had a mean particle size of 330-400 nm while the ζ potential varied from 36-39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size comparable to that determined by dynamic light scattering. ISLP/p2CMVmIL-12 complexes prepared using free-base NEACE-T:DOPE (1/2) at charge ratios of 3/1 and 5/1 (±) provided the highest levels of transgene expression, 18 times more than the levels provided by the salt-form. Secreted levels of mIL-12 p70 were 75 times higher for ISLP/p2CMVmIL-12 complexes than naked p2CMVmIL-12 and nearly 4 times higher than PEI 25 kDa/p2CMVmIL-12 complexes. Conclusions. The transfection efficiency of the ISLPs was dependent on the site of cholesterol conjugation, amount of colipid, and charge ratio. The highest levels of transgene expression were provided by NEACE-T:DOPE (1/2)/p2CMVmIL-12 at a 3/1 (±) charge ratio.",
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T1 - Novel water insoluble lipoparticulates for gene delivery

AU - Furgeson, D. Y.

AU - Cohen, R. N.

AU - Mahato, Ram I

AU - Kim, S. W.

PY - 2002/4/29

Y1 - 2002/4/29

N2 - Purpose. The objective was to design and prepare water insoluble lipoparticulates (ISLPs) for efficient gene delivery to lung tissue. Methods. Nona{(ethylenimine)-co-[(2-aminoethyl)-N-choleseteryloxycarbonyl- ethylenimine]} (NEACE-T) was synthesized in both its free-base and chloride salt-forms using linear polyethylenimine (PEI, Mw 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitelechelic Nα-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by 1H-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a secondary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a colipid, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1/1, 1/2, and 2/1 molar ratios and the lipoparticulates were hydrated and filtered. ISLP/p2CMVmIL-12 complexes were characterized for particle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Results. Transgene expression was dependent on the site of cholesterol conjugation, lipononamer:colipid molar ratio, and ISLP/p2CMVmIL-12 charge ratios. ISLP/p2CMVmIL-12 complexes were nontoxic to murine colon adenocarcinoma (CT-26) cells at 9/1 (±) or lower, had a mean particle size of 330-400 nm while the ζ potential varied from 36-39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size comparable to that determined by dynamic light scattering. ISLP/p2CMVmIL-12 complexes prepared using free-base NEACE-T:DOPE (1/2) at charge ratios of 3/1 and 5/1 (±) provided the highest levels of transgene expression, 18 times more than the levels provided by the salt-form. Secreted levels of mIL-12 p70 were 75 times higher for ISLP/p2CMVmIL-12 complexes than naked p2CMVmIL-12 and nearly 4 times higher than PEI 25 kDa/p2CMVmIL-12 complexes. Conclusions. The transfection efficiency of the ISLPs was dependent on the site of cholesterol conjugation, amount of colipid, and charge ratio. The highest levels of transgene expression were provided by NEACE-T:DOPE (1/2)/p2CMVmIL-12 at a 3/1 (±) charge ratio.

AB - Purpose. The objective was to design and prepare water insoluble lipoparticulates (ISLPs) for efficient gene delivery to lung tissue. Methods. Nona{(ethylenimine)-co-[(2-aminoethyl)-N-choleseteryloxycarbonyl- ethylenimine]} (NEACE-T) was synthesized in both its free-base and chloride salt-forms using linear polyethylenimine (PEI, Mw 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitelechelic Nα-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by 1H-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a secondary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a colipid, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1/1, 1/2, and 2/1 molar ratios and the lipoparticulates were hydrated and filtered. ISLP/p2CMVmIL-12 complexes were characterized for particle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Results. Transgene expression was dependent on the site of cholesterol conjugation, lipononamer:colipid molar ratio, and ISLP/p2CMVmIL-12 charge ratios. ISLP/p2CMVmIL-12 complexes were nontoxic to murine colon adenocarcinoma (CT-26) cells at 9/1 (±) or lower, had a mean particle size of 330-400 nm while the ζ potential varied from 36-39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size comparable to that determined by dynamic light scattering. ISLP/p2CMVmIL-12 complexes prepared using free-base NEACE-T:DOPE (1/2) at charge ratios of 3/1 and 5/1 (±) provided the highest levels of transgene expression, 18 times more than the levels provided by the salt-form. Secreted levels of mIL-12 p70 were 75 times higher for ISLP/p2CMVmIL-12 complexes than naked p2CMVmIL-12 and nearly 4 times higher than PEI 25 kDa/p2CMVmIL-12 complexes. Conclusions. The transfection efficiency of the ISLPs was dependent on the site of cholesterol conjugation, amount of colipid, and charge ratio. The highest levels of transgene expression were provided by NEACE-T:DOPE (1/2)/p2CMVmIL-12 at a 3/1 (±) charge ratio.

KW - Cytokine

KW - Insoluble

KW - Lipononamer

KW - Lipoparticulates

KW - Transgene

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