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Chondroitin Sulfate-Coated DNA-Nanoplexes Enhance Transfection Efficiency by Controlling Plasmid Release from Endosomes: A New Insight into Modulating Nonviral Gene Transfection
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
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2015 (English)In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 25, no 25, 3907-3915 p.Article in journal (Refereed) Published
Abstract [en]

Degradation of plasmid DNA (pDNA) in the endosome compartment and its release to the cytosol are the major hurdles for efficient gene transfection. This is generally addressed by using transfection reagents that can overcome these limitations. In this article, the first report is presented which suggests that controlling the release of pDNA from endosome is the key for achieving efficient transfection. In this study, chondroitin sulfate (CS)-coated DNA-nanoplexes are developed using a modular approach where CS is coated post-pDNA/PEI nanoplex formation. To ensure good stability of the nanoplexes, imine/enamine chemistry is exploited by reacting aldehyde-modified chondroitin sulfate (CS-CHO) with free amines of pDNA/PEI complex. This supramolecular nanocarrier system displays efficient cellular uptake, and controlled endosomal pDNA release without eliciting any cytotoxicity. On the contrary, burst release of pDNA from endosome (using chloroqine) results in significant reduction in gene expression. Unlike pDNA/PEI-based transfection, the nanoparticle design presented here shows exceptional stability and gene transfection efficiency in different cell lines such as human colorectal cancer cells (HCT116), human embryonic kidney cells (HEK293), and mouse skin-derived mesenchymal stem cells (MSCs) using luciferase protein as a reporter gene. This new insight will be valuable in designing next generation of transfection reagents.

Place, publisher, year, edition, pages
2015. Vol. 25, no 25, 3907-3915 p.
Keyword [en]
chondroitin sulfate, endosomal escape, gene delivery, mesenchymal stem cells, nanoparticles
National Category
Physical Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-259095DOI: 10.1002/adfm.201500695ISI: 000357268900013OAI: oai:DiVA.org:uu-259095DiVA: diva2:843463
Funder
EU, FP7, Seventh Framework Programme
Available from: 2015-07-28 Created: 2015-07-27 Last updated: 2017-12-04Bibliographically approved
In thesis
1. New insights into principles of scaffolds design for bone application
Open this publication in new window or tab >>New insights into principles of scaffolds design for bone application
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules.

Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7.

During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence.  The different inflammatory responses to HA hydrogels were observed (Appendix).

Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo.

Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model.

Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over a long term at a closer physiological level. Yet, efficient gene delivery reagents are needed. In paper IV, two novel gene delivery nanoplexes were developed by post coating DNA-nanoplexes with chondroitin sulfate (CS). To ensure the stability, aldehyde-modified CS (CS-CHO) reacted with free amines of pDNA/PEI complexes. We provided first evidence that CS-CHO coated nanoplexes controlled the release from endosomes, which is essential for higher transfection efficiency.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 87 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1459
Keyword
Chondroitin sulfate, hyaluronic acid, pH, cross-linking chemistry, bone morphogenetic protein, lithium, mesenchymal stem cell, in vivo.
National Category
Medical Materials
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-308318 (URN)978-91-554-9767-5 (ISBN)
Public defence
2017-01-17, Room 80121, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2016-12-20 Created: 2016-11-24 Last updated: 2016-12-20

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Yan, HongjiPodiyan, OommenYu, DiHilborn, JönsVarghese, Oommen P.

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