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The nanoparticles in the size range of 200–500 nm were prepared and the presence of poly(ethylene glycol) chains on the surface was confirmed by Electron spectroscopy for chemical analysis

Long-circulating poly(ethylene glycol)-modified gelatin nanoparticles for intracellular delivery.

Pharmaceutical research, no. 7 (2002): 1061-1067

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摘要

The objective of this study was to develop and characterize long-circulating, biodegradable, and biocompatible nanoparticulate formulation as an intracellular delivery vehicle.Poly(ethylene glycol) (PEG)-modified gelatin was synthesized by reacting Type-B gelatin with PEG-epoxide. The nanoparticles, prepared by pH and temperature controll...更多

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简介
  • With advances in molecular and cell biology, there are increasing numbers of opportunities for cell-specific targeting and delivery of drugs [1,2,3,4,5,6,7].
  • For. DNA delivery, cationic lipid complexes, and cationic polymer complexes have been studied extensively for somatic gene therapy [8,9,10].
  • DNA delivery, cationic lipid complexes, and cationic polymer complexes have been studied extensively for somatic gene therapy [8,9,10]
  • These vector systems, suffer from poor efficiency of transfection, lack of selectivity, and in some cases, poor stability of the DNA after intracellular delivery [11,12].
  • Being a hydrophobic slow degrading polymer that results in highly acidic local microenvironment, PLGA is not a very effective polymer matrix for DNA encapsulation [17,18]
重点内容
  • With advances in molecular and cell biology, there are increasing numbers of opportunities for cell-specific targeting and delivery of drugs [1,2,3,4,5,6,7]
  • The percentage of free amine groups of gelatin that were reacted by poly(ethylene glycol) (PEG)-epoxide in the modified gelatin derivative was determined by trinitrobenzenesulfonic acid (TNBS) assay as a function of the amount of PEG-epoxide added per gram of gelatin
  • The 30% PEG-modified derivative was subsequently used for preparation of PEGylated gelatin nanoparticles
  • Immortalized BT-20 cells were grown in 96 well plates at an initial seeding density of 10,000 cells per well in 200 ␮l of supplemented Eagle’s minimum essential media (MEM)
  • The nanoparticles in the size range of 200–500 nm were prepared and the presence of PEG chains on the surface was confirmed by Electron spectroscopy for chemical analysis (ESCA)
  • The results of this study are very encouraging for the development of an intracellular delivery system for drugs and genes that can offer long-circulating property, is efficiently internalized by cells and remains intact in the endosomes and lysosomes
方法
  • Materials

    Type-B gelatin (225 bloom strength) with 100–115 millimoles of free carboxylic acid per 100 g of protein, an isoelectric point of 4.7–5.2, and an average molecular weight 40,000– Kaul and Amiji

    50,000 daltons was purchased from Sigma Chemical Company (St Louis, Missouri).
  • Monomethoxy-poly(ethylene glycol) (PEG) with a molecular weight of 5,000 daltons was obtained from Fluka Chemika/Biochemika (Ronkonkoma, New York).
  • The BT-20 estrogen receptor negative human breast carcinoma cell line was obtained from American Type Culture Collection (ATCC, Rockville, Maryland) and maintained in Eagle’s minimum essential media (MEM) supplemented with 10 mM pyruvate, non-essential amino acids, L-glutamine, and 10% fetal bovine serum at 37°C and 5% CO2 atmosphere.
  • All aqueous solutions were prepared exclusively in deionized distilled water (Nanopure II, Barnstead/Thermolyne, Dubuque, Iowa)
结果
  • The percentage of free amine groups of gelatin that were reacted by PEG-epoxide in the modified gelatin derivative was determined by TNBS assay as a function of the amount of PEG-epoxide added per gram of gelatin.
  • When 0.5 g of PEG-epoxide was added per gram of gelatin, percent PEGylation, as determined by TNBS assay was approximately around 30%.
  • ESCA is a surface sensitive technique because only those electrons that leave the surface without energy loss will contribute to the peak signifying that element.
  • Those electrons originating from far below the surface (>100 Angstroms) suffer energy loss through collisions and are unable to make it out of the surface, or they escape the surface with considerable energy loss [23]
结论
  • PEG-modified (PEGylated) gelatin derivative was synthesized for preparation of long-circulating nanoparticles with capacity for intracellular delivery of drugs and genes.
  • Cell uptake studies of the control and PEGylated gelatin nanoparticles confirmed that they were internalized by endocytotic pathway and remained stable during the vesicular transport process.
  • The results of this study are very encouraging for the development of an intracellular delivery system for drugs and genes that can offer long-circulating property, is efficiently internalized by cells and remains intact in the endosomes and lysosomes
总结
  • Introduction:

    With advances in molecular and cell biology, there are increasing numbers of opportunities for cell-specific targeting and delivery of drugs [1,2,3,4,5,6,7].
  • For. DNA delivery, cationic lipid complexes, and cationic polymer complexes have been studied extensively for somatic gene therapy [8,9,10].
  • DNA delivery, cationic lipid complexes, and cationic polymer complexes have been studied extensively for somatic gene therapy [8,9,10]
  • These vector systems, suffer from poor efficiency of transfection, lack of selectivity, and in some cases, poor stability of the DNA after intracellular delivery [11,12].
  • Being a hydrophobic slow degrading polymer that results in highly acidic local microenvironment, PLGA is not a very effective polymer matrix for DNA encapsulation [17,18]
  • Objectives:

    The objective of this study was to develop and characterize long-circulating, biodegradable, and biocompatible nanoparticulate formulation as an intracellular delivery vehicle.
  • Methods:

    Materials

    Type-B gelatin (225 bloom strength) with 100–115 millimoles of free carboxylic acid per 100 g of protein, an isoelectric point of 4.7–5.2, and an average molecular weight 40,000– Kaul and Amiji

    50,000 daltons was purchased from Sigma Chemical Company (St Louis, Missouri).
  • Monomethoxy-poly(ethylene glycol) (PEG) with a molecular weight of 5,000 daltons was obtained from Fluka Chemika/Biochemika (Ronkonkoma, New York).
  • The BT-20 estrogen receptor negative human breast carcinoma cell line was obtained from American Type Culture Collection (ATCC, Rockville, Maryland) and maintained in Eagle’s minimum essential media (MEM) supplemented with 10 mM pyruvate, non-essential amino acids, L-glutamine, and 10% fetal bovine serum at 37°C and 5% CO2 atmosphere.
  • All aqueous solutions were prepared exclusively in deionized distilled water (Nanopure II, Barnstead/Thermolyne, Dubuque, Iowa)
  • Results:

    The percentage of free amine groups of gelatin that were reacted by PEG-epoxide in the modified gelatin derivative was determined by TNBS assay as a function of the amount of PEG-epoxide added per gram of gelatin.
  • When 0.5 g of PEG-epoxide was added per gram of gelatin, percent PEGylation, as determined by TNBS assay was approximately around 30%.
  • ESCA is a surface sensitive technique because only those electrons that leave the surface without energy loss will contribute to the peak signifying that element.
  • Those electrons originating from far below the surface (>100 Angstroms) suffer energy loss through collisions and are unable to make it out of the surface, or they escape the surface with considerable energy loss [23]
  • Conclusion:

    PEG-modified (PEGylated) gelatin derivative was synthesized for preparation of long-circulating nanoparticles with capacity for intracellular delivery of drugs and genes.
  • Cell uptake studies of the control and PEGylated gelatin nanoparticles confirmed that they were internalized by endocytotic pathway and remained stable during the vesicular transport process.
  • The results of this study are very encouraging for the development of an intracellular delivery system for drugs and genes that can offer long-circulating property, is efficiently internalized by cells and remains intact in the endosomes and lysosomes
表格
  • Table1: High Resolution C1s Peak Analysis of ESCA on Surfaces of Gelatin and PEGylated Gelatin Nanoparticlesa
Download tables as Excel
基金
  • This study was partially supported by the Northeastern University Research and Development Grant
  • ESCA was performed at the NESAC/BIO, University of Washington, Seattle, WA, which is supported by NIH grant RR-01296
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