Engineered polyallylamine nanoparticles for efficient in vitro transfection.
OBJECTIVE
Cationic polymers (i.e. polyallylamine, poly-L-lysine) having primary amino groups are poor transfection agents and possess high cytotoxicity index when used without any chemical modification and usually entail specific receptor mediated endocytosis or lysosomotropic agents to execute efficient gene delivery. In this report, primary amino groups of polyallylamine (PAA, 17 kDa) were substituted with imidazolyl functions, which are presumed to enhance endosomal release, and thus enhance its gene delivery efficiency and eliminate the requirement of external lysosomotropic agents. Further, systems were cross-linked with polyethylene glycol (PEG) to prepare PAA-IAA-PEG (PIP) nanoparticles and evaluated them in various model cell lines.


METHODS
The efficacy of PIP nanoparticles in delivering a plasmid encoding enhanced green fluorescent protein (EGFP) gene was assessed in COS-1, N2a and HEK293 cell lines, while their cytotoxicity was investigated in COS-1 and HEK293 cell lines. The PAA was chemically modified using imidazolyl moieties and ionically cross-linked with PEG to engineer nanoparticles. The extent of substitution was determined by ninhydrin method. The PIP nanoparticles were further characterized by measuring the particle size (dynamic light scattering and transmission electron microscopy), surface charge (zeta potential), DNA accessibility and buffering capacity. The cytotoxicity was examined using the MTT method.


RESULTS
In vitro transfection efficiency of synthesized nanoparticles is increased up to several folds compared to native polymer even in the presence of serum, while maintaining the cell viability over 100% in COS-1 cells. Nanoparticles possess positive zeta potential between 5.6-13 mV and size range of 185-230 nm in water. The accessibility experiment demonstrated that nanoparticles with higher degree of imidazolyl substitution formed relatively loose complexes with DNA. An acid-base titration showed enhanced buffering capacity of modified PAA.


CONCLUSIONS
The PIP nanoparticles reveal tremendous potential as novel delivery system for achieving improved transfection efficiency, while keeping the cells at ease.
Pathak
Atul
A
Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110007, India.
Aggarwal
Anita
A
Kurupati
Raj K
RK
Patnaik
Soma
S
Swami
Archana
A
Singh
Yogendra
Y
Kumar
Pradeep
P
Vyas
Suresh P
SP
Gupta
Kailash C
KC
D000818:Animals; D002021:Buffers; D019556:COS Cells; D002234:Carbodiimides / Q000737:chemistry; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004996:Ethidium / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011073:Polyamines / Q000737:chemistry* / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000494:pharmacology; D044967:Serum / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013499:Surface Properties; D014162:Transfection / Q000379:methods*
D002021:Buffers; D002234:Carbodiimides; D011073:Polyamines; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D049452:Green Fluorescent Proteins; C063994:polyallylamine; D011092:Polyethylene Glycols; D004247:DNA; D004996:Ethidium