Polymeric Nanoparticles Containing Conjugated Phospholipase A2 for Nonviral Gene Delivery

Le, Huong Thu; Rao, Gururaj; Hirko, Aaron C.; Hughes, Jeffrey A.

doi:10.1021/mp900192p

Cited by 5 publications

(6 citation statements)

References 38 publications

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“…PLC was never used for non‐viral gene delivery systems, phospholipases originating from insect venoms have been applied for this purpose before. [ 18 , 19 ]…”

Section: Introductionmentioning

confidence: 99%

“…[17] While L. m. PLC was never used for non-viral gene delivery systems, phospholipases originating from insect venoms have been applied for this purpose before. [18,19] Once inside the cytoplasm, the DNA cargo must be transported toward and into the nucleus for transcription. This process is hindered by several hurdles, such as the crowded nature of the cytosol and the nuclear pore, a highly regulated barrier that only allows selected substrates to pass through.…”

Section: Introductionmentioning

confidence: 99%

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The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent

2022

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Non‐viral gene delivery agents, such as cationic lipids, polymers, and peptides, mainly rely on charge‐based and hydrophobic interactions for the condensation of DNA molecules into nanoparticles. The human protein mitochondrial transcription factor A (TFAM), on the other hand, has evolved to form nanoparticles with DNA through highly specific protein‐protein and protein‐DNA interactions. Here, the properties of TFAM are repurposed to create a DNA transfection agent by means of protein engineering. TFAM is covalently fused to Listeria monocytogenes phospholipase C (PLC), an enzyme that lyses lipid membranes under acidic conditions, to enable endosomal escape and human vaccinia‐related kinase 1 (VRK1), which is intended to protect the DNA from cytoplasmic defense mechanisms. The TFAM/DNA complexes (TFAMoplexes) are stabilized by cysteine point mutations introduced rationally in the TFAM homodimerization site, resulting in particles, which show maximal activity when formed in 80% serum and transfect HeLa cells in vitro after 30 min of incubation under challenging cell culture conditions. The herein developed TFAM‐based DNA scaffolds combine interesting characteristics in an easy‐to‐use system and can be readily expanded with further protein factors. This makes the TFAMoplex a promising tool in protein‐based gene delivery.

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“…PLC was never used for non‐viral gene delivery systems, phospholipases originating from insect venoms have been applied for this purpose before. [ 18 , 19 ]…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent

2022

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“…Dopamine (DA) can be oxidized and polymerized by oxygen in the air in an alkaline environment, forming a thin surface-adhered polydopamine (PDA) film on the surface of various inorganic and organic materials through the self-polymerization process. [23][24][25] PDA can be easily covalently bound to molecular rich in amino and sulfhydryl groups in a weak alkaline environment. Therefore, PDA coating can not only significantly improve the hydrophilicity and biological properties of materials, but also provide active functional groups for further surface modification of materials.…”

Section: Introductionmentioning

confidence: 99%

Preparation of PEI‐modified nanoparticles by dopamine self‐polymerization for efficient DNA delivery

Liu

Yang

Liu

et al. 2022

Biotech and App Biochem

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Achieving efficient and safe gene delivery is of great significance to promote the development of gene therapy. In this work, a polydopamine (PDA) layer was coated on the surface of Fe3O4 nanoparticles (NPs) by dopamine (DA) self‐polymerization, and then magnetic Fe3O4 NPs were prepared by the Michael addition between amino groups in polyethyleneimine (PEI) and PDA. The prepared Fe3O4 NPs (named Fe3O4@PDA@PEI) were characterized by Fourier transform infrared (FTIR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). As an efficient and safe gene carrier, the potential of Fe3O4@PDA@PEI was evaluated by agarose gel electrophoresis, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, fluorescence microscopy, and flow cytometry. The results show that the Fe3O4@PDA@PEI NPs are stable hydrophilic NPs with a particle size of 50–150 nm. It can efficiently condense DNA at low N/P ratios and protect it from nuclease degradation. In addition, the Fe3O4@PDA@PEI NPs have higher safety than PEI. Further, the Fe3O4@PDA@PEI/DNA polyplexes could be effectively absorbed by cells and successfully transfected and exhibit higher cellular uptake and gene transfection efficiency than PEI/DNA polyplexes. The findings indicate that the Fe3O4@PDA@PEI NPs have the potential to be developed into a novel gene vector.

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“…Bee venom PLA2 has been used in previous studies in attempts to improve nonviral delivery of DNA to cells in vitro [393][394][395] . Our initial attempts to prepare PLA2 for DNA delivery involved attaching the polyacridine peptide (Acr-Lys) 6 -Cys to the lysines of PLA2 through the heterobifunctional crosslinker GMBS (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Another study covalently linked bee venom PLA2 to PEI and used it to transfect cells in vitro. PEI with PLA2 was less toxic than PEI without PLA2, and was able to produce higher levels of transgene expression at high N:P ratios 394 . PLA2 was delivered to cells in vitro with DNA in cationic cycloamylose nanogels, and showed higher transgene expression than nanogel and DNA alone 395 .…”

Section: Introductionmentioning

confidence: 94%

Nonviral gene delivery to the liver

Crowley¹

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Diseases of the liver have a large impact on human health. Genetic disorders, metabolic disorders, alcoholism, cancer, or infections can all impair liver function. If serious enough, a liver transplant may be necessary, a major surgical procedure which requires lifelong immune suppression and relies on the availability of donor livers. Gene therapy is being intensively studied as a potential method to treat many disorders, including disorders of the liver. While viral gene therapy has seen some success, possible side effects make it risky, so nonviral gene delivery vectors are being developed. Unfortunately, these nonviral vectors do not yet have the efficiency of the viral vectors. Nonviral gene delivery vectors face many challenges in vivo. The vectors must protect DNA from nucleases while it moves through the bloodstream, they must avoid nonspecific uptake, they must be enter the correct cells, and must enter the nucleus before the DNA can be expressed. If any step of this process fails, there will be very little, if any, expression, and it may be impossible to determine what went wrong. One impediment to nonviral gene delivery research is the transition from in vitro studies to in vivo studies. The cancer derived cell lines most often used for in vitro transfections are rapidly dividing, which makes nuclear entry much easier than in the whole animal. While primary cells would be a more accurate model of the in vivo environment, the number of cells that can be obtained from tissues is small, and primary cells usually cannot be cultured for long. This limits the number of experiments that can be done with each preparation of cells. To overcome this, we have miniaturized transfection assays, including the transfection of mouse primary hepatocytes with luciferase in 384 well plates. Because fewer cells are needed, more experiments can be performed with each liver preparation. Another issue introduced by the differences between in vitro and in vivo research is circulatory stability. In vitro, large particles with strong positive charges are desired, because they sink down onto the cells and are attracted to the negatively charged cellular membranes. However, in vivo these particles will aggregate serum proteins and become lodged in narrow capillary beds in the lungs or other organs, often causing toxicity. While this behavior can usually be overcome through PEGylation, improving a particle's circulatory half-life will still improve its chances of finding the correct target. Scavenger receptors found on liver nonparenchymal cells are very efficient at removing negatively charged particles from the bloodstream. We have shown that dosing large amounts of PEGylated polyacridine DNA polyplex can saturate the scavenger receptors and improve circulatory half-life. We have also shown that large doses of PEGylated peptide, with or without acridine groups, can inhibit scavenger receptor uptake through the formation of peptide-protein nanoparticles. By inhibiting scavenger receptor uptake, DNA can be successfully hydrodynami...

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Polymeric Nanoparticles Containing Conjugated Phospholipase A2 for Nonviral Gene Delivery

Cited by 5 publications

References 38 publications

The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent

The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent

Preparation of PEI‐modified nanoparticles by dopamine self‐polymerization for efficient DNA delivery

Nonviral gene delivery to the liver

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