Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan-dextran sulfate nanoparticles enhances their expression and anti-cancer effects

Our plain-English summary of Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan-dextran sulfate nanoparticles enhances their expression and anti-cancer effects, by V. Perumal, F. Arfuso, Y. Chen, et al (Molecular and Cellular Biochemistry, 2018).

The study

• Authors: V. Perumal, F. Arfuso, Y. Chen, S. Fox, A.M. Dharmarajan
• Journal: Molecular and Cellular Biochemistry
• Published: 2018;443(1-2):205-213
• Cited by: 6 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan-dextran (CS-DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS-DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300 nm for empty particles to 337 nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25 mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS-DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE® HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS-DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.

In plain English

This was a laboratory (in vitro) study in two human malignant pleural mesothelioma cell lines, JU77 and ONE58. The authors made chitosan–dextran sulfate (CS–DS) nanoparticles by complex coacervation carrying plasmid DNA for full-length secreted frizzled-related protein 4 (SFRP4), its cysteine-rich domain (CRD), its netrin-like domain (NLD), or an empty green fluorescent protein vector; results were set against conventional FUGENE HD transfection, largely as reported in the authors' earlier experiments. Particle sizes ran from 300.2 ± 17.2 nm (empty) to 337.0 ± 6.2 nm (CRD), zeta potentials were around 25 mV or above, and DNA entrapment efficiency was 74–89% across the four constructs. Cells received 40 ng of encapsulated DNA per well; maximum gene expression occurred at 48 h, whereas in the earlier FUGENE HD experiments maximal expression was seen only after about 6 days. In both cell lines SFRP4 and the NLD reduced cell viability (MTT, 48 h), while the CRD gave no effect or slight stimulation. The authors described a minimal 2% effect on cell morphology and viability for the EGFP nanoparticles, and a greater effect of 45% on morphology and anti-proliferation for the NLD compared with the CRD. SFRP4- and NLD-treated cells showed GFP-associated vesicular membrane structures. The authors concluded the approach shows promise as a non-viral gene delivery system.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory experiment: preparation and physical characterisation of chitosan–dextran sulfate nanoparticles carrying plasmid DNA, then transfection of two mesothelioma cell lines with imaging and MTT viability assays
• Who was studied: Two human malignant mesothelioma cell lines, JU77 and ONE58, originally derived from pleural effusions of different patients with malignant pleural mesothelioma; experiments done in triplicate in three independent biological experiments (Curtin University, Perth, Australia)
• Exposure: Not an asbestos exposure study: cells were treated with chitosan–dextran sulfate nanoparticles encapsulating plasmid DNA for SFRP4, the CRD, the NLD, or empty pEGFP N1 vector, 40 ng DNA per well, compared with FUGENE HD transfection at 100 ng per well
• Main results:
  • Nanoparticle characterisation: empty particles 300.2 ± 17.2 nm, SFRP4 318.5 ± 6.6 nm, CRD 337.0 ± 6.2 nm, NLD 325.0 ± 5.6 nm, pEGFP N1 328.5 ± 9.0 nm; all zeta potentials around 25 mV or above
  • DNA entrapment efficiency was 74 to 89% across the four DNA constructs
  • With nanoparticle delivery, maximum gene expression occurred at 48 h in both cell lines; in the authors' earlier FUGENE HD experiments maximal GFP expression was seen only after approximately 6 days
  • In both JU77 and ONE58 cells, SFRP4 and the NLD nanoparticles reduced cell viability, while the CRD gave no effect or slight stimulation (MTT at 48 h, n = 3, p less than 0.05)
  • The CS–DS/EGFP nanoparticles showed a minimal 2% effect on cell morphology and viability compared with CS–DS/SFRP4 and its domains, while the CS–DS/SFRP4 NLD exerted a greater effect of 45% on cell morphology and anti-proliferative effect compared with the CRD
  • Cells transfected with nanoparticle-delivered SFRP4 and NLD showed distinctive vesicular membrane structures associated with GFP fluorescence
• The authors' conclusion: The authors concluded that the chitosan–dextran sulfate formulation kept DNA integrity and entrapment efficiency high, that biological effects of SFRP4/domain gene delivery appeared earlier and with less DNA than with a conventional transfection reagent, and that chitosan SFRP4 and NLD nanoparticle gene delivery shows promise as a non-viral delivery system
• Limitations the authors state:
  • Higher nanoparticle concentrations were toxic to the cultures, which limited the amount of DNA that could be delivered
  • The mechanism of the earlier and lower-dose biological effect is unknown to the authors
  • The authors present the work as preliminary non-viral in vitro data intended as a basis for possible future animal and preclinical studies

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Malignant Mesothelioma.

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