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  • 1. Backly, R. E.
    et al.
    Todeschi, M. R.
    Varghese, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Cancedda, R.
    Mastrogiacomo, M.
    Host cell recruitment patterns by BMP-2 releasing hyaluronic acid gels in a mouse subcutaneous model2014In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 8, p. 65-65Article in journal (Other academic)
  • 2. Barman, Jharna
    et al.
    Gurav, Deepanjali
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen Podiyan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    2 '-N-Guanidino, 4 '-C-ethylene bridged thymidine (GENA-T) modified oligonucleotide exhibits triplex formation with excellent enzymatic stability2015In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 5, no 16, p. 12257-12260Article in journal (Refereed)
    Abstract [en]

    Here we present the synthesis and characterization of a new 2'-N-Guanidino, 4'-C-ethylene bridged thymidine (GENA-T) modified oligonucleotide possessing North-locked sugar conformation. Incorporation of GENA-T nucleotide though did not change the thermal stability of the oligonucleotides toward the complementary RNA; it significantly increased the stability of the parallel triplex at pH 7. The melting temperature of the triplex was increased by +9.5 degrees C as compared to that of the isosequential unmodified sequence. Moreover this modification imparted exceptional nuclease stability to the oligonucleotides for over 33 h. This study clearly demonstrates that GENA-T modified oligonucleotides could improve triplex formation with phenomenal enzymatic stability and could be used for various biomedical applications.

  • 3.
    Bermejo, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Kadekar, Sandeep
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Tavares da Costa, Marcus Vinicius
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Mechanics.
    Podiyan, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gamstedt, E. Kristofer
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Mechanics.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    First Aldol-Crosslinked Hyaluronic Acid Hydrogel: Fast and Hydrolytically Stable Gel with Tissue Adhesive PropertiesIn: Chemical Sciences Journal, ISSN 2150-3494Article in journal (Refereed)
    Abstract [en]

    Currently, there are limited approaches to tailor 3D scaffolds crosslinked with a stable covalent C-C bond that does not require any catalysts or initiators. We present here the first hydrogels employing aldol condensation chemistry that exhibit exceptional physicochemical properties. We investigated the aldol-crosslinking chemistry using two types of aldehyde-modified hyaluronic acid (HA) derivatives, namely; an enolizable HA-aldehyde (HA-Eal) and a non-enolizable HA-aldehyde (HA-Nal). Hydrogels formed using HA-Eal demonstrate inferior crosslinking efficiency (due to intramolecular loop formation), when compared with hydrogels formed by mixing HA-Eal and HA-NaI leading to a cross-aldol product. The change in mechanical properties as a result of crosslinking at different pH is determined using rheological measurements and is interpreted in terms of molecular weight between cross-links (Mc). The novel HA cross-aldol hydrogels demonstrate excellent hydrolytic stability and favorable mechanical properties but allow hyaluronidase mediated enzymatic degradation. Interestingly, residual aldehyde functionality within the aldol product leads to adhesion to tissue as demonstrated by bonding two bone tissues. The aldehyde functionality also permits facile post-synthetic modifications with nucleophilic reagents such as Alexa FluorTM 488. Finally, we demonstrate that the novel hydrogel is biocompatible with encapsulated stem cells that show a linear rate of expansion in our 3–6 days of study.

  • 4.
    Bermejo-Velasco, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Azémar, Alice
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Bioengineering and Nanomedicine Lab, Faculty of Medicine and Health Technologies and BioMediTech Institute, Tampere University, Korkeakoulunkatu 3, Tampere 33720, Finland.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Modulating thiol pKa promotes disulfide formation at physiological pH: An elegant strategy to design disulfide cross-linked hyaluronic acid hydrogels2019In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 20, no 3, p. 1412-1420Article in journal (Refereed)
    Abstract [en]

    The disulfide bond plays a crucial role in protein biology and has been exploited by scientists to develop antibody-drug conjugates, sensors and for the immobilization other biomolecules to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants and long reaction times. We demonstrate here that thiol-substrates containing electron-withdrawing groups at the β-position influence the deprotonation of the thiol group, which is the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction kinetics using small molecule substrate such as L-cysteine indicated disulfide formation at a 2.8-fold higher (k1 = 5.04 x 10-4 min-1) reaction rate as compared to the conventional thiol substrate, namely 3-mercaptopropionic acid (k1 = 1.80 x 10-4 min-1) at physiological pH (pH 7.4). Interestingly, the same effect could not be observed when N-acetyl-L-cysteine substrate (k1 = 0.51 x 10-4 min-1) was used. We further grafted such thiol-containing molecules (cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely hyaluronic acid (HA) and determined the pKa value of different thiol groups by spectrophotometric analysis. The electron-withdrawing group at the β-position reduced the pKa of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-ActCys) and 8.1 for HA-thiol (HA-SH) derivatives respectively. These experiments further confirmed that the concentration of thiolate (R-S-) ions could be increased with the presence of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed hydrogels within 3.5 minutes or 10 hours, respectively at pH 7.4. After completion of crosslinking reaction both gels demonstrated a storage modulus G’ ≈3300–3500 Pa, indicating comparable levels of crosslinking. The HA-SH gel, on the other hand, did not form any gel at pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and reductive degradation). We believe our study provides a valuable insight on the factors governing the disulfide formation and our results are useful to develop strategies that would facilitate generation of stable thiol functionalized biomolecules or promote fast thiol oxidation according to the biomedical needs.

  • 5.
    Bermejo-Velasco, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Bioengineering and Nanomedicine Lab, Faculty of Biomedical Sciences and Engineering, Tampere University of Technology, and BioMediTech Institute, 33720, Tampere, Finland.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Thiazolidine chemistry revisited: a fast, efficient and stable click-type reaction at physiological pH2018In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 54, no 88, p. 12507-12510Article in journal (Refereed)
    Abstract [en]

    We describe the fast reaction kinetics between 1,2-aminothiols and aldehydes. Under physiological conditions such a click-type reaction afforded a thiazolidine product that remains stable and did not require any catalyst. This type of bioorthogonal reaction offers enormous potential for the coupling of biomolecules in an efficient and biocompatible manner.

  • 6.
    Chatterjee, S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Pathmasiri, W.
    Plashkevych, O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Honcharenko, D.
    Varghese, O.P.
    Maiti, M.
    Chattopadhyaya, J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The chemical nature of the 2'-substituent in the pentose-sugar dictates the pseudoaromatic character of the nucleobase (pKa) in DNA/RNA.2006In: Org. Biomol. Chem., Vol. 4, p. 1675-1686Article in journal (Refereed)
    Abstract
  • 7.
    Chatterjee, Subhrangsu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Pathmasiri, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Honcharenko, D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Varghese, O.P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Maiti, M
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    The chemical nature of the 2'-substituent in the pentosesugar dictates the pseudoaromatic character of the nucleobase (pKa) in DNA/RNA2006In: Org Biomol Chem, Vol. 4, p. 1675-1686Article in journal (Refereed)
  • 8. Ferreira, Silvia A
    et al.
    Motwani, Meghna S
    Faull, Peter A
    Seymour, Alexis J
    Yu, Tracy T L
    Enayati, Marjan
    Taheem, Dheraj K
    Salzlechner, Christoph
    Haghighi, Tabasom
    Kania, Ewa M
    Oommen, Oommen P
    Ahmed, Tarek
    Loaiza, Sandra
    Parzych, Katarzyna
    Dazzi, Francesco
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Festy, Frederic
    Grigoriadis, Agamemnon E
    Auner, Holger W
    Snijders, Ambrosius P
    Bozec, Laurent
    Gentleman, Eileen
    Bi-directional cell-pericellular matrix interactions direct stem cell fate2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, no 1, article id 4049Article in journal (Refereed)
    Abstract [en]

    Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells' 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells' reciprocal interactions with their pericellular environment when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their surroundings by synthesizing, secreting and arranging proteins pericellularly or by degrading the hydrogel. hMSC's interactions with this local environment have a role in regulating hMSC fate, with a secreted proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate.

  • 9.
    Gurav, Deepanjali
    et al.
    Savitri Bai Phule Pune Univ, Dept Chem, Pune, Maharashtra, India..
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Hamad, Osama A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen Podiyan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Chondroitin sulfate coated gold nanoparticles: a new strategy to resolve multidrug resistance and thromboinflammation2016In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 52, no 5, p. 966-969Article in journal (Refereed)
    Abstract [en]

    We have developed the first chondroitin sulfate polymer coated gold nanoparticles that can simultaneously overcome mulidrug resistance in cancer cells and suppress thromboinflammation triggered by the chemotherapeutic drug.

  • 10.
    Han, Yuanyuan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Qiu, Zhen
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Tian, Bo
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics. Tech Univ Denmark, Dept Micro & Nanotechnol, DK-2800 Kongens Lyngby, Denmark.
    Leifer, Klaus
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification2019In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 127, p. 188-193Article in journal (Refereed)
    Abstract [en]

    DNA technology based bio-responsive nanomaterials have been widely studied as promising tools for biomedical applications. Gold nanoparticles (AuNPs) and graphene oxide (GO) sheets are representative zero- and two-dimensional nanomaterials that have long been combined with DNA technology for point-of-care diagnostics. Herein, a cascade amplification system based on duplex-specific nuclease (DSN)-assisted target recycling and electrocatalytic water-splitting is demonstrated for the detection of microRNA. Target microRNAs can form DNA: RNA heteroduplexes with DNA probes on the surface of AuNPs, which can be hydrolyzed by DSN. MicroRNAs are preserved during the reaction and released into the suspension for the digestion of multiple DNA probes. After the DSN-based reaction, AuNPs are collected and mixed with GO to form AuNP/GO nanocomposite on an electrode for the following electrocatalytic amplification. The utilization of AuNP/GO nanocomposite offers large surface area, exceptional affinity to water molecules, and facilitated mass diffusion for the water-splitting reaction. For let-7b detection, the proposed biosensor achieved a limit detection of 1.5 fM in 80 min with a linear detection range of approximately four orders of magnitude. Moreover, it has the capability of discriminating non-target microRNAs containing even single-nucleotide mismatches, thus holding considerable potential for clinical diagnostics.

  • 11.
    Hilborn, Jöns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Jeronimo, B.
    Keynote: Safe and efficient in-vivo gene gene transfer and silencing technologies using natural pathways2012In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, Vol. 6, no suppl 1, p. 190-190Article in journal (Other academic)
    Abstract [en]

    Introduction: The advance in nucleic acids therapeutic had been outstanding in recent years, which have opened new possibilities in regenerative medicine to tackle many serious diseases. To make nucleic acids based therapy a reality, the fundamental problem of tissue specific cellular delivery has to be accomplished. Here we present a new approach to develop the next generation of delivery vectors.

    Materials and methods: Hyaluronic acid were modified to complex DNA plasmids for luciferase transfection. Transfection: HCT 116 cells and mice where tranfected by adding HA-DNA complex. The transfection experiment with commercially available reagent polyethyleneimine (PEI) and plasmid alone were used as controls.

    Results and discussion: Efficient tranfection was shown using the new non toxic and safe non viral vector. Using ECM components (such as hyaluronic acid or HA) that allow both in vitroandin vivo transfection of plasmid DNA in CD44 positive cells. The chemically modified ECM components (non-toxic) binds to nucleic acids and are taken in by cells by natural receptor mediated endocytosis. The amount transported via these routes naturally is around 10 gram per day to give a potential capacity far exceeding the need for transfection if it is targeted. By molecular association strategies (joining the bandwagon) it is now possible for nucleic acids to follow these pathways that utilise the highly efficient receptor mediated endocytosis- door opener into cells.

  • 12.
    Honcharenko, D.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Varghese, O.P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Plashkevych, O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Barman, J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Synthesis and Structure of Novel Conformationally-constrained 1'2'-Azetidine-Fused Bicyclic Pyrimidine Nucleosides: Their Incorporation into Oligo-DNAs and the Thermal Stability of the Heteroduplexes2006In: Journal of Organic Chemistry, Vol. 71, p. 299-314Article in journal (Refereed)
  • 13.
    Honcharenko, Dmytro
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Barman, Jharna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, p. 5635-5646Article in journal (Refereed)
    Abstract [en]

    The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing NorthEast conformationally constrained 1‘,2‘-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2‘,4‘-ethylene-bridged (aza-ENA-T) nucleoside, and 2‘-alkoxy modified nucleosides (2‘-O-Me-T and 2‘-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (ΔTm = 2.5−4 °C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4−5.5 °C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of 5−6 °C per modification. The 2‘-O-Me-T and 2‘-O-MOE-T modifications, on the other hand, showed an increased of Tm by 0.5 °C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (1.4−2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (1.7−8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (1.1−3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2‘-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2‘-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3‘-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3‘-exonuclease preferentially hydrolyzed the 3‘-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3‘-exonuclease cleaved at the 3‘-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5‘-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5‘-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2‘-O-Me-T, and 2‘-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.

  • 14.
    Kadekar, Sandeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Karlsson, Kira
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Ålander, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Podiyan, Oommen
    Bioengineering and Nanomedicine Lab, Faculty of Biomedical Sciences and Engineering, Tampere University of Technology, and BioMediTech Institute, 33720, Tampere, Finland.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Synthetic design of asymmetric miRNA with engineered 3′-overhang to improve strand selection2019In: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 16, p. 597-604Article in journal (Refereed)
    Abstract [en]

    We have developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3′-end by adding extra nucleotides. Addition of seven nucleotides at the 3′-ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two-ends, which resulted in selective RISC recruitment as demonstrated by the stem-loop quantitative PCR experiment. Such selective recruitment was also corroborated at the protein level by Western blot analysis. In order to investigate the functional effect due to selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that the recruitment of miR strand is responsible for inducing apoptosis as well as to inhibit invasiveness of cancer cells. Recruitment of miR* strand, on the other hand, showed opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNA to enhance their therapeutic potential.

  • 15.
    Kisiel, Marta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Ventura, M.
    George, Anu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Critical Assessment of rhBMP-2 Mediated Bone Induction: an In Vitro and In Vivo Evaluation2012In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, Vol. 6, no SI, p. 39-39Article in journal (Other academic)
  • 16.
    Kisiel, Marta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ventura, Manuela
    Podiyan, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    George, Anu
    Walboomers, X Frank
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Varghese, Oommen P
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Critical assessment of rhBMP-2 mediated bone induction: An in vitro and in vivo evaluation.2012In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 162, no 3, p. 646-653Article in journal (Refereed)
    Abstract [en]

    Understanding the influence of formulation and storage conditions on rhBMP-2 bioactivity is extremely important for its clinical application. Reports in the literature show that different research groups employ different parameters such as formulation conditions, storage, doses for in vivo applications etc. that makes it difficult to correlate results from different experiments. We therefore decided to rationalize these anomalies by performing a basic study on such parameters using two commercially available BMPs. Our in vitro experiments suggest that BMPs from different sources have significant differences in their bioactivity. The clinically approved rhBMP-2 (InductOs®; BMP-P) showed superior stability, compared to rhBMP-2 from R&D Systems (BMP-R) at physiological pH (determined by ALP assay). This BMP-P also showed lower binding to polypropylene Eppendorf tube. The BMP-R almost lost its bioactivity within 30min at physiological pH and also shows more adhesion to plastic surfaces. This aggregation behavior was unequivocally ascertained by performing light scattering studies of the two BMPs, which revealed linear aggregation with time for BMP-R unlike BMP-P. The in vitro results were also reflected in the in vivo experiments, in a rat ectopic model with injectable hyaluronic acid (HA) hydrogel as BMP carrier. After 7weeks post-implantation we observed larger bone volume with oriented collagen in the BMP-P group but a smaller bone with disoriented collagen in the BMP-R case. Our results highlight the large difference in activity between seemingly identical substances and also the importance of proper handling of such sensitive proteins.

  • 17. Martinez-Sanz, Elena
    et al.
    Ossipov, Dmitri A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Larsson, Sune
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Jonsson, Kenneth B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Bone reservoir: Injectable hyaluronic acid hydrogel for minimal invasive bone augmentation2011In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 152, no 2, p. 232-240Article in journal (Refereed)
    Abstract [en]

    A strategy has been designed to develop hyaluronic acid (HA) hydrogel for in vivo bone augmentation using minimal invasive technique. A mild synthetic procedure was developed to prepare aldehyde modified HA by incorporating an amino-glycerol side chain via amidation reaction and selective oxidation of the pendent group. This modification, upon mixing with hydrazide modified HA formed hydrazone-crosslinked hydrogel within 30 s that was stable at physiological pH. In vitro experiments showed no cytotoxicity of hydrogel with the controlled release of active bone morphogenic protein-2 (BMP-2). In vivo evaluation of this gel as a BMP-2 carrier was performed by injecting gels over the rat calvarium and showed bone formation in 8 weeks in correlation with the amount of BMP-2 loaded (0, 1 and 30 pig) within the gel. Furthermore, hydrogels with 30 Kg of BMP-2 induced less bone formation upon subcutaneous injection in comparison with subperiosteal implantation. Histological examination showed newly formed bone with a high expression of osteocalcin, osteopontin and with angiogenic bone marrow when higher BMP-2 concentration was employed. Our result suggests that novel HA hydrogels could be used as a BMP-2 carrier and can promote bone augmentation for potential orthopedic applications.

  • 18.
    Martinez-Sanz, Elena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kisiel, Marta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Engstrand, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Reich, Karoline
    Bohner, Marc
    Jonsson, Kenneth B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Köhler, Thomas
    Müller, Ralph
    Ossipov, Dmitri
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Minimally invasive mandibular bone augmentation using injectable hydrogels2012In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, Vol. 6, no S3, p. s15-s23Article in journal (Refereed)
    Abstract [en]

    Hyaluronic acid-based hydrogels are proven biocompatible materials and excellent carriers of bone morphogenetic protein-2 (BMP-2) that have been successfully tested for bone generation in vivo. Different formulations, with or without nanohydroxyapatite, have shown promise for craniofacial applications. In this study, 28 rats were used to investigate whether it is possible to achieve mandibular bone augmentation upon injection of novel hyaluronic acid-based hydrogels containing nanohydroxyapatite and different concentrations of BMP-2 (0, 5 and 150ÎŒg/ml). The biomaterials were injected subperiosteally through fine needles into the innate mandibular diastema, imitating a clinical procedure for resorbed mandibles. No incisions, flaps or sutures were necessary. After 8weeks the mandibles were evaluated by peripheral quantitative computed tomography (pQCT), micro-computed tomography (ÎŒCT), histology, immunohistochemistry and fluorochrome labelling. As a result, engineered bone was observed in all treated mandibles, with a statistically significant increase in mandibular bone volume correlated with the amount of BMP-2 loaded in the hydrogel formula. We therefore demonstrated that minimally invasive mandibular bone augmentation is possible upon injection in rats, when using the appropriate injectable scaffolds. This represents an attractive clinical alternative for oral implantology patients.

  • 19.
    Mittapelli, Lavanya L.
    et al.
    Univ Mumbai, Dept Chem, Mumbai 400098, Maharashtra, India.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Gholap, Sachin P.
    Indian Inst Technol, Dept Chem, Mumbai 400076, Maharashtra, India.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Gore, Kiran R.
    Univ Mumbai, Dept Chem, Mumbai 400098, Maharashtra, India.
    A turn-on fluorescent GFP chromophore analog for highly selective and efficient detection of H2S in aqueous and in living cells2019In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 298, article id 126875Article in journal (Refereed)
    Abstract [en]

    Hydrogen sulphide is a gaseous neurotransmitter responsible for neuronal function and controls vast range of physiological functions. Herein, we report the synthesis and evaluation of novel Green Fluorescent Protein (GFP) chromophore analog, acryloyl-4-(p-hydroxybenzylidene)-5-imidazolidinone (AHBI) for turn-on fluorescent detection of H2S over wide range of anions and various biologically important competitive thiols. AHBI probe exhibited high selectivity and sensitivity, high fluorescence stability, large stokes shift and lower detection limit (15.85 ppb) for H2S in complete water medium. Cell imaging studies in human colon cancer cells (HCT116) and normal human dermal fibroblasts (HDF) confirmed the compatibility and versatility of AHBI probe at micromolar level. Overall, we believe the AHBI, as an optical probe will be useful to investigate the role of H2S in various physiological processes, regulation of cancer cell growth, and in pathogenic events.

  • 20.
    Nawale, Ganesh N.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Bahadorikhalili, Saeed
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Sengupta, Pallabi
    Bose Inst, Dept Biophys, Biomol NMR & Drug Design Lab, P-1-12 CIT Scheme 7M, Kolkata, India.
    Kadekar, Sandeep
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Chatterjee, Subhrangsu
    Bose Inst, Dept Biophys, Biomol NMR & Drug Design Lab, P-1-12 CIT Scheme 7M, Kolkata, India.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    4 '-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading2019In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 55, no 62, p. 9112-9115Article in journal (Refereed)
    Abstract [en]

    We designed novel 4 '-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in approximate to 4.9-fold more efficient knockdown than unmodified siRNA.

  • 21.
    Oommen, Oommen P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Wang, Shujiang
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Kisiel, Marta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Sloff, Marije
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Smart Design of Stable Extracellular Matrix Mimetic Hydrogel: Synthesis, Characterization, and In Vitro and In Vivo Evaluation for Tissue Engineering2013In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 23, no 10, p. 1273-1280Article in journal (Refereed)
    Abstract [en]

    The simplicity and versatility of hydrazone crosslinking has made it a strategy of choice for the conjugation of bioactive molecules. However, the labile nature of hydrazone linkages and reversibility of this coupling reaction restricts its full potential. Based on the fundamental understanding of hydrazone stability, this problem is circumvented by resonance-stabilization of a developing N2 positive charge in a hydrazone bond. A novel chemistry is presented to develop a resilient hydrazone bond that is stable and non- reversible under physiological conditions. A carbodihydrazide (CDH) type hydrazide derivative of the biomolecule forms intrinsically stabilized hydrazone-linkages that are nearly 15-fold more stable at pH 5 than conventional hydrazone. This chemoselective coupling reaction is catalyst-free, instantaneous, and virtually non-cleavable under physiological conditions, therefore can serve as a catalyst-free alternative to click chemistry. This novel crosslinking reaction is used to tailor a hyaluronan hydrogel, which delivered exceptional hydrolytic stability, mechanical properties, low swelling, and controlled enzymatic degradation. These desired characteristics are achieved without increasing the chemical crosslinking. The in vivo evaluation of this hydrogel revealed neo-bone with highly ordered collagen matrix mimicking natural bone regeneration. The proximity ligation assay or PLA is used to detect blood vessels, which highlighted the quality of engineered tissue.

  • 22.
    Ossipov, Dmitri A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Piskounova, Sonya
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Functionalization of Hyaluronic Acid with Chemoselective Groups via a Disulfide-Based Protection Strategy for In Situ Formation of Mechanically Stable Hydrogels2010In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 11, no 9, p. 2247-2254Article in journal (Refereed)
    Abstract [en]

    Functionalization of hyaluronic acid (HA) with chemoselective groups enables in situ (in vivo) formation of HA-based materials in minimally invasive injectable manner. Current methods of HA modification with such groups primarily rely on the use of a large excess of a reagent to introduce a unique reactive handle into HA and, therefore, are difficult to control. We have developed the new protective group strategy based on initial mild cleavage of a disulfide bond followed by elimination of the generated 2-thioethoxycarbonyl moiety ultimately affording free amine-type functionality, such as hydrazide, aminooxy, and carbazate. Specifically, new modifying homobifunctional reagents have been synthesized that contain a new divalent disulfide-based protecting group. Amidation of HA with these reagents gives rise to either one-end coupling product or to intra/intermolecular cross-linking of the HA chains. However, after subsequent treatment of the amidation reaction mixture with dithiothreitol (DTT), these cross-linkages are cleaved, ultimately exposing free amine-type groups. The same methodology was applied to graft serine residues to the HA backbone, which were subsequently oxidized into aldehyde groups. The strategy therefore encompasses a new approach for mild and highly controlled functionalization of HA with both nucleophilic and electrophilic chemoselective functionalities with the emphasis for the subsequent conjugation and in situ cross-linking. A series of new hydrogel materials were prepared by mixing the new HA-aldehyde derivative with different HA-nucleophile counterparts. Rheological properties of the formed hydrogels were determined and related to the structural characteristics of the gel networks. Human dermal fibroblasts remained viable while cultured with the hydrogels for 3 days, with no sign of cytotoxicity, suggesting that the gels described in this study are candidates for use as growth factors delivery vehicles for tissue engineering applications.

  • 23.
    Ossipov, Dmitri A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Yang, Xia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Varghese, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Kootala, Sujit
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Modular approach to functional hyaluronic acid hydrogels using orthogonal chemical reactions2010In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 46, no 44, p. 8368-8370Article in journal (Refereed)
    Abstract [en]

    A modular approach for the synthesis of hyaluronic acid hydrogels using orthogonal chemoselective reactions for subsequent enzymatic decomposition to nanoparticles is described.

  • 24.
    Paidikondala, Maruthibabu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Kadekar, Sandeep
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Innovative strategy for 3D transfection of primary human stem cells with BMP-2 expressing plasmid DNA: A clinically translatable strategy for ex vivogene therapy2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 1, article id 56Article in journal (Refereed)
    Abstract [en]

    Ex vivo gene therapy offers enormous potential for cell-based therapies, however, cumbersome in vitro cell culture conditions have limited its use in clinical practice. We have optimized an innovative strategy for the transient transfection of bone morphogenetic protein-2 (BMP-2) expressing plasmids in suspended human stem cells within 5-min that enables efficient loading of the transfected cells into a 3D hydrogel system. Such a short incubation time for lipid-based DNA nanoparticles (lipoplexes) reduces cytotoxicity and at the same time reduces the processing time for cells to be transplanted. The encapsulated human mesenchymal stromal/stem cells (hMSCs) transfected with BMP-2 plasmid demonstrated high expression of an osteogenic transcription factor, namely RUNX2, but not the chondrogenic factor (SOX9), within the first three days. This activation was also reflected in the 7-day and 21-day experiment, which clearly indicated the induction of osteogenesis but not chondrogenesis. We believe our transient transfection method demonstrated in primary MSCs can be adapted for other therapeutic genes for different cell-based therapeutic applications.

  • 25.
    Paidikondala, Maruthibabu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel2019In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 20, no 3, p. 1317-1324Article in journal (Refereed)
    Abstract [en]

    Small interfering RNAs (siRNAs) are powerful toolsfor post-transcriptional gene silencing, which offers enormousopportunities for tissue engineering applications. However, poorserum stability, inefficient intracellular delivery, and inevitabletoxicity of transfection reagents are the key barriers for their clinicaltranslation. Thus, innovative strategies that allow safe and efficientintracellular delivery of the nucleic acid drugs at the desired site isurgently needed for a smooth clinical translation of therapeuticallyappealing siRNA-based technology. In this regard, we havedeveloped an innovative siRNA transfection protocol that employsa short incubation time of just 5 min. This allows easy transfection insuspension followed by transplantation of the cells in a hyaluronicacid (HA) hydrogel system. We also report here the unique ability ofsiRNA to bind HA that was quantified by siRNA release andrheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA insuspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to beessential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with orwithout removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removalof excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improvedknockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) asdetermined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the proteinlevels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allowsminimal manipulation of cells that are to be transplanted and reduce toxicity.

  • 26.
    Paidikondala, Maruthibabu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    New insight on siRNA transfection in three dimensions: Improved gene silencing in human mesenchymal stem cells encapsulated in hyaluronicacid hydrogelManuscript (preprint) (Other academic)
  • 27.
    Paidikondala, Maruthibabu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Rangasami, Vignesh Kumar
    Tampere Univ, Fac Med & Hlth Technol, Bioengn & Nanomed Lab, Korkeakoulunkatu 3, Tampere 33720, Finland;Tampere Univ, BioMediTech Inst, Korkeakoulunkatu 3, Tampere 33720, Finland.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Casalini, Tommaso
    SUPSI, Dept Innovat Technol, Inst Mech Engn & Mat Engn, CH-6928 Manno, Switzerland;Swiss Fed Inst Technol, Dept Chem & Appl Biosci, Inst Chem & Bioengn, CH-8093 Zurich, Switzerland.
    Perale, Giuseppe
    SUPSI, Dept Innovat Technol, Inst Mech Engn & Mat Engn, CH-6928 Manno, Switzerland.
    Kadekar, Sandeep
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Mohanty, Gaurav
    Tampere Univ, Fac Engn & Nat Sci, Mat Sci & Environm Engn, Tampere, Finland.
    Salminen, Turkka
    Tampere Univ, Tampere Microscopy Ctr, Tampere, Finland.
    Oommen, Oommen P.
    Tampere Univ, Fac Med & Hlth Technol, Bioengn & Nanomed Lab, Korkeakoulunkatu 3, Tampere 33720, Finland;Tampere Univ, BioMediTech Inst, Korkeakoulunkatu 3, Tampere 33720, Finland.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method2019In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 58, no 9, p. 2815-2819Article in journal (Refereed)
    Abstract [en]

    Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular-matrix-derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size- and concentration-dependent gene silencing in a CD44-positive human osteosarcoma cell line (MG-63) and in human mesenchymal stromal cells (hMSCs). This native HA-based siRNA transfection represents the first report on an anionic, non-viral delivery method that resulted in approximately 60% gene knockdown in both cell types tested, which correlated with a reduction in translation levels.

  • 28.
    Paidikondala, Maruthibabu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Rangasami, Vignesh Kumar
    Tampere University of Technology, Finland.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Casalini, Tommaso
    Perale, Giuseppe
    Podiyan, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hyaluronic acid facilitates non-cationic siRNA delivery and gene silencing in CD44 positive cellsManuscript (preprint) (Other academic)
  • 29.
    Paidikondala, Maruthibabu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Wang, Shujiang
    Maisonneuve-Rosemont Hospital Research Centre & Department of Ophthalmology, University of Montreal.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Sune
    Department of Surgical Sciences, Section of Orthopedics, Uppsala University Hospital.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Impact of Hydrogel Cross-Linking Chemistry on the in Vitro and in VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-22019In: ACS Applied Bio Materials, ISSN 2576-6422Article in journal (Refereed)
    Abstract [en]

    Designing strategies to deliver functional proteins at physiologically relevant concentrations using chemically cross-linked biocompatible hydrogels is a major field of research. However, the impact of cross-linking chemistry on the encapsulated protein bioactivity is rarely studied. Here we examine the two well-known cross-linking reactions namely; hydrazone cross-linking chemistry and thiol-Michael addition reaction to form hyaluronic acid (HA) hydrogels. As a therapeutic protein, we employed recombinant human bone morphogenetic protein-2 (rhBMP-2) for this study. Incubation of rhBMP-2 with HA functionalized with a thiol diminished phosphorylation of Smad 1/5/8, a signal transducer for osteogenic differntiation, whereas an aldehyde functionalized HA had no effect. This indicates that thiol functionalized polymers indeed has an impact on protein function. To validate this result in an in vivo setting we performed BMP-2 induced bone formation in a rat ectopic model. These experiments revealed that the hydrazone-cross-linked HA-hydrogel induced significantly higher bone formation (18.90 ± 4.25 mm3) as compared to the HA-thiol-Michael hydrogels (1.25 ± 0.52 mm3) after 8 weeks as determined by micro-computed tomography. The histological examination of the neo-bone indicated that hydrazone-hydrogels promoted a better quality of bone formation with improved mineralization and collagen formation as compared to the thiol-Michael hydrogels. We believe such a direct comparison of two cross-linking chemistries will provide new insight for developing biomaterials for protein delivery for in vivo applications.

  • 30.
    Paidikondala, Maruthibabu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Wang, Shujiang
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Yan, Hongji
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Podiyan, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Sune
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Rational Design of Biomaterials for Growth Factor Delivery: Impact of Hydrogel Crosslinking Chemistry on the In Vitroand In VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-2Manuscript (preprint) (Other academic)
  • 31.
    Podiyan, Oommen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Garousi, Javad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Sloff, Marije
    Varghese, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Tailored Doxorubicin-Hyaluronan Conjugate as a Potent Anticancer Glyco-Drug: An Alternative to Prodrug Approach2014In: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, Vol. 14, no 3, p. 327-333Article in journal (Refereed)
    Abstract [en]

    Releasibility of doxorubicin from drug-conjugates is believed to be a prerequisite for its anti- cancer activity. Here, a new glyco-drug approach that circumvents the releasibility restriction is reported, opening a new possibility to design efficient, target specific drug delivery system. It is discovered that stable amide coupling of doxorubicin (DOX) tohyaluronan (HA) shows dose dependent cytotoxicity to CD44 positive human coloncancer cells (HCT116) as compared to human breast cancer cells(MCF-7) and mouse fibroblast cells (NIH-3T3), which express less CD44 receptor. This direct conjugation approach is an easy scalable strategy that could be adopted to design innocuous anti-tumor nanoparticle formulations.

  • 32.
    Podiyan, Oommen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Wang, Shujiang
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Kisiel, Marta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Smart design of stable hydrazone crosslinked extracellular matrix mimetic hydrogel for tissue engineering application2012In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, Vol. 6, no suppl 1, p. 192-192Article in journal (Other academic)
    Abstract [en]

    Injectable hydrogels are important biomaterials with enormous applications. They are used for various biomedical applications such as diagnostics, 3D cell culture matrix, drug reservoir, encapsulation of bioactive compounds and growth factors, scaffold for tissue engineering etc. We here present our recent development in our efforts to develop hydrogel scaffolds with enhanced rigidity, stability, swelling characteristics. Hydrazone crosslinked gels are attractive due to its simplicity and versatility which could be formed by mixing appropriate aldehyde and hydrazide functionalized hyaluronan. By fine-tuning the electronic character around the hydrazone linkage, we succeeded in developing extremely stable hydrazone bond and utilized it for developing hyaluronan (HA) based synthetic extracellular matrix (ECM) hydrogel. Among the different hydrazides tested, we identified carbonyldihydrazide (CDH) as the best candidate to deliver stable hydrazone linkage. This stability is presumably due to extensive delocalization of the positive charge across neighboring amino groups of CDH. The hydrolytic stability imparted by this group was found to be several folds under acidic, basic and physiological pH when compared to other hydrazones. This tailored hydrogel with CDH also exhibited superior swelling and mechanical properties and enzymatic stability which makes it ideal for tissue engineering application.

  • 33. Rahman, Cheryl V.
    et al.
    Kuhn, Gisela
    White, Lisa J.
    Kirby, Giles T. S.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    McLaren, Jane S.
    Cox, Helen C.
    Rose, Felicity R. A. J.
    Mueller, Ralph
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Shakesheff, Kevin M.
    PLGA/PEG-hydrogel composite scaffolds with controllable mechanical properties2013In: Journal of Biomedical Materials Research. Part B - Applied biomaterials, ISSN 1552-4973, E-ISSN 1552-4981, Vol. 101B, no 4, p. 648-655Article in journal (Refereed)
    Abstract [en]

    Biodegradable polymer scaffolds have great potential for regenerative medicine applications such as the repair of musculoskeletal tissues. Here, we describe the development of scaffolds that blend hydrogel components with thermoplastic materials, combining the unique properties of both components to create mouldable formulations. This study focuses on the structural and mechanical properties of the composite scaffolds, produced by combining temperature-sensitive poly(DL-lactic acid-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) particles with a hydrogel component [Pluronic F127, fibrin or hyaluronic acid (HyA)]. The composite formulations solidified over time at 37 degrees C, with a significant increase (p 0.05) in compressive strength observed from 15 min to 2 h at this temperature. The maximum compressive strength was 1.2 MPa for PLGA/PEG-Pluronic F127 scaffolds, 2.4 MPa for PLGA/PEG-HyA scaffolds and 0.6 MPa for PLGA/PEG-fibrin scaffolds. Porosity for each of the PLGA/PEG-hydrogel formulations tested was between 50 and 51%. This study illustrates the ability to combine this thermoplastic PLGA/PEG system with hydrogels to fabricate composite scaffolds, and demonstrates that altering the particle to hydrogel ratio produces scaffolds with varying mechanical properties.

  • 34.
    Roshanbinfar, Kaveh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Friedrich-Alexander-Universitaet Erlangen-Nuernberg (FAU), Erlangen, Germany.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Tampere University of Technology, Tampere, Finland.
    Injectable and thermoresponsive pericardial matrix derived conductive scaffold for cardiac tissue engineering2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 51, p. 31980-31988Article in journal (Refereed)
    Abstract [en]

    Scaffolds derived from decellularized cardiac tissue offer an enormous advantage for cardiac applications as they recapitulate biophysical and cardiac specific cues. However, poor electrical conductivity and mechanical properties severely compromise the therapeutic potential of these matrices. Dispersion of multiwall carbon nanotubes (MWCTs) in these scaffolds could improve their mechanical and electrical properties. However, the inherent hydrophobicity and poor dispersibility of these materials under aqueous conditions limit their outcome. We have developed a modified MWCNT functionalized with carbodihydrazide (CDH) residues that significantly improved their dispersibility and suppressed cytotoxicity in HL-1 cardiomyocytes. We found that the doping of CDH functionalized MWCNT (CDH-MWCNT) as low as 0.5 wt% to the pericardial matrix hydrogel (PMNT) induced the necessary electrical conductivity and significantly improved the mechanical properties of the hydrogel. Cardiomyocytes cultured on a PMNT scaffold triggered proliferation and significantly increased the expression of cardiac-specific gap junction protein, namely connexin 43. Such a phenomenon was not observed when cardiomyocytes were cultured on the pericardial matrix derived gels without MWCNT or on gelatin-fibronectin coated 2D cultures. The PMNT gels displayed excellent biophysical characteristics resulting in the clustering of cardiomyocytes with synchronous contraction, which is crucial for the successful integration to the host tissue.

  • 35.
    Todeschi, Maria R.
    et al.
    Univ Genoa, Dept Expt Med DIMES, Genoa, Italy.;IRCCS Policlin San Martino, Genoa, Italy..
    El Backly, Rania M.
    Univ Genoa, Dept Expt Med DIMES, Genoa, Italy.;IRCCS Policlin San Martino, Genoa, Italy.;Alexandria Univ, Conservat Dent Dept, Endodont, Fac Dent, Alexandria, Egypt..
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Cancedda, Ranieri
    Univ Genoa, Dept Expt Med DIMES, Genoa, Italy.;IRCCS Policlin San Martino, Genoa, Italy..
    Mastrogiacomo, Maddalena
    Univ Genoa, Dept Expt Med DIMES, Genoa, Italy.;IRCCS Policlin San Martino, Genoa, Italy..
    Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment2017In: Regenerative Medicine, ISSN 1746-0751, E-ISSN 1746-076X, Vol. 12, no 5, p. 525-539Article in journal (Refereed)
    Abstract [en]

    Aim: This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Materials & methods: Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Results & conclusion: Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

  • 36.
    Varghese, Oommen P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Barman, Jharna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Pathmasiri, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Honcharenko, Dmytro
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Thymidine (Aza-ENA-T): Synthesis, Structure, Physical, and Biochemical Studies of Aza-ENA-T-Modified Oligonucleotides2006In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 47, p. 15173-15187Article in journal (Refereed)
    Abstract [en]

    The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn- protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 < P < 27 degrees, 44 degrees < phi(m) < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with E-a 25.4 kcal mol(-1), which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a T m increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.

  • 37.
    Varghese, Oommen P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Kisiel, Marta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Martinez-Sanz, Elena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Ossipov, Dmitri A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Synthesis of Guanidinium-Modified Hyaluronic Acid Hydrogel2010In: Macromolecular rapid communications, ISSN 1022-1336, E-ISSN 1521-3927, Vol. 31, no 13, p. 1175-1180Article in journal (Refereed)
    Abstract [en]

    In this report, a new guanidinylating reagent is presented, which was developed without any protection/deprotection strategy and was successfully employed for linking to hyaluronan in aqueous solution. The dually functionalised HA biopolymer bearing guanidinium and hydrazide groups was synthesised to form hydrogel in Ho less than a minute when mixed with aldehyde-modified HA. This hydrogel exhibited higher storage modulus with enhanced stability in PBS when compared to the non-guanidine-containing gel. The gel shift assay showed that this biopolymer formed a stable complex with DNA as well as efficient gene transfection to cells that express HA-receptor CD44. The toxicity studies of this polymer with fibroblast cells revealed that the cells were almost 80% viable after 4 d of incubation at high HA concentration (2.5 x 10(-3) m).

  • 38.
    Varghese, Oommen P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Podiyan, Oommen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Wang, S
    Kisiel, Marta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Injectable hyaluronic acid hydrogel for bone tissue engineering2012In: European Cells and Materials, ISSN 1473-2262, E-ISSN 1473-2262, Vol. 23, no supplement 3, p. 2-Article in journal (Refereed)
  • 39.
    Varghese, Oommen P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Sun, Weilun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Ossipov, Dmitri A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    In Situ Cross-Linkable High Molecular Weight Hyaluronan−Bisphosphonate Conjugate for Localized Delivery and Cell-Specific Targeting: A Hydrogel Linked Prodrug Approach2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 25, p. 8781-8783Article in journal (Refereed)
    Abstract [en]

    We present here a novel synthesis route to functionalize high molecular weight hyaluronan (HMW-HA) with a hydrazide group and a bioactive ligand, namely bisphosphonate (BP). For this purpose, a new symmetrical self-immolative biscarbazate linker has been devised. The hydrazide group was used to form hydrazone cross-linked hydrogel upon treating with previously described aldehyde modified hyaluronan. The 1:1 weight ratio of these two polymers gave hydrogel in less than 30 s. In this communication we present the first in vitro results showing that even though HA can target CD44 positive cancer cells (HCT-116), receptor mediated endocytosis could only occur by cleavage of high molecular weight HA with an ubiquitous enzyme, hyaluronidase (Hase). The cancer cells are known to overexpress CD44 receptors and also increase the hyaluronidase activity in vivo. Thus the pro-drug design, based on drug conjugation to HMW-HA, represents a new drug delivery platform where the drug potency is triggered by Hase mediated degradation of the HA-drug conjugate. We have successfully demonstrated that the cross-linkable HA−BP conjugate first undergoes Hase-mediated scission to the fragments of suitable sizes so as to be internalized by CD44 positive cells. The specificity of this targeting was proven by comparing the results with less CD44 positive HEK-293T cells. The localized delivery of such drugs at the surgical resection site opens up avenues to control tumor recurrence after removal of the tumor. In the form of hydrogel it would prevent systemic exposure of the drug and would allow its controlled release.

  • 40.
    Wang, Shujiang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Gurav, Deepanjali
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen Podiyan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Insights into the Mechanism and Catalysis of Oxime Coupling Chemistry at Physiological pH2015In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 21, no 15, p. 5980-5985Article in journal (Refereed)
    Abstract [en]

    The dynamic covalent-coupling reaction involving alpha-effect nucleophiles has revolutionized bioconjugation approaches, due to its ease and high efficiency. Key to its success is the discovery of aniline as a nucleophilic catalyst, which made this reaction feasible under physiological conditions. Aniline however, is not so effective for keto substrates. Here, we investigate the mechanism of aniline activation in the oxime reaction with aldehyde and keto substrates. We also present carboxylates as activating agents that can promote the oxime reaction with both aldehyde and keto substrates at physiological pH. This rate enhancement circumvents the influence of alpha-effect by forming H-bonds with the rate-limiting intermediate, which drives the reaction to completion. The combination of aniline and carboxylates had a synergistic effect, resulting in a similar to 14-31-fold increase in reaction rate at pD 7.4 with keto substrates. The biocompatibility and efficiency of carboxylate as an activating agent is demonstrated by performing cell-surface oxime labeling at physiological pH using acetate, which showed promising results that were comparable with aniline.

  • 41.
    Wang, Shujiang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Kadekar, Sandeep
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Tampere Univ Technol, Bioengn & Nanomed Lab, Fac Biomed Sci & Engn, Tampere 33720, Finland.;BioMediTech Inst, Tampere 33720, Finland..
    Jena, Naresh K.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Chakraborty, Sudip
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 2193Article in journal (Refereed)
    Abstract [en]

    We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.

  • 42.
    Wang, Shujiang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Kadekar, Sandeep
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Tampere University of Technology, and BioMediTech Institute, Finland.
    Jena, Naresh K.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Chakraborty, Sudip
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Saline catalyse oxime reaction at physiological pH: overcoming a major limitation of bioorthogonal reactionIn: Article in journal (Refereed)
    Abstract [en]

    We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most bio-friendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.

  • 43.
    Wang, Shujiang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. Univ Montreal, Maisonneuve Rosemont Hosp Res Ctr, Montreal, PQ, Canada;Univ Montreal, Dept Ophthalmol, Montreal, PQ, Canada.
    Nawale, Ganesh N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Tampere Univ, Fac Med & Hlth Technol, Bioengn & Nanomed Lab, Korkeakoulunkatu 3, Tampere 33720, Finland;Tampere Univ, BioMediTech Inst, Korkeakoulunkatu 3, Tampere 33720, Finland.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Influence of ions to modulate hydrazone and oxime reaction kinetics to obtain dynamically cross-linked hyaluronic acid hydrogels2019In: Polymer Chemistry, ISSN 1759-9954, E-ISSN 1759-9962, Vol. 10, no 31, p. 4322-4327Article in journal (Refereed)
    Abstract [en]

    Dynamic covalent chemistry forming hydrazone and oxime linkages is attractive due to its simplicity, selectivity and compatibility under aqueous conditions. However, the low reaction rate at physiological pH hampers its use in biomedical applications. Herein, we present different monovalent and bivalent aqueous salt solutions as bio-friendly, non-toxic catalysts which can drive the hydrazone and oxime reactions with excellent efficacy at physiological pH. Direct comparison of hydrazone and oxime reactions using a small molecule model, without any salt catalysis, indicated that oxime formation is 6-times faster than hydrazone formation. Addition of different salts (NaCl, NaBr, KCl, LiCl, LiClO4, Na2SO4, MgCl2 and CaCl2) accelerated the pseudo-first-order reaction kinetics by similar to 1.2-4.9-fold for acylhydrazone formation and by similar to 1.5-6.9-fold for oxime formation, in a concentration-dependent manner. We further explored the potential of such catalysts to develop acylhydrazone and oxime cross-linked hyaluronic acid (HA) hydrogels with different physicochemical properties without changing the degree of chemical modification. Analogous to the small molecule model system, the addition of monovalent and divalent salts as catalysts significantly reduced the gelling time. The gelling time for the acylhydrazone cross-linked HA-hydrogel (1.6 wt%) could be reduced from 300 min to 1.2 min by adding 100 mM CaCl2, while that for the oxime cross-linked HA-hydrogel (1.2 wt%) could be reduced from 68 min to 1.1 min by adding 50 mM CaCl2. This difference in the gelling time also resulted in hydrogels with differential swelling properties as measured after 24 h. Our results are the first to demonstrate the use of salts, for catalyzing hydrogel formation under physiologically relevant conditions.

  • 44.
    Wang, Shujiang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Yan, Hongji
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Mild and Efficient Strategy for Site-Selective Aldehyde Modification of Glycosaminoglycans: Tailoring Hydrogels with Tunable Release of Growth Factor2013In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, no 7, p. 2427-2432Article in journal (Refereed)
    Abstract [en]

    Aldehydes have been used as an important bioorthogonal chemical reporter for conjugation of large polymers and bioactive substances. However, generating aldehyde functionality on carbohydrate-based biopolymers without changing its native chemical structure has always persisted as a challenging task. The common methods employed to achieve this require harsh reaction conditions, which often compromise the structural integrity and biological function of these sensitive molecules. Here we report a mild and simple method to graft aldehydes groups on glycosaminoglycans (GAGs) in a site-selective manner without compromising the structural integrity of the biopolymer. This regio-selective modification was achieved by conjugating the amino-glycerol moiety on the carboxylate residue of the polymer, which allowed selective cleavage of pendent diol groups without interfering with the C2C3 diol groups of the native glucopyranose residue. Kinetic evaluation of this reaction demonstrated significant differences in second-order reaction rate for periodate oxidation (by four-orders of magnitude) between the two types of vicinal diols. We employed this chemistry to develop aldehyde modifications of sulfated and nonsulfated GAGs such as hyaluronic acid (HA), heparin (HP), and chondroitin sulfate (CS). We further utilized these aldehyde grafted GAGs to tailor extracellular matrix mimetic injectable hydrogels and evaluated its rheological properties. The composition of the hydrogels was also found to modulate release of therapeutic protein such as FGF-2, demonstrating controlled release (60%) for over 14 days. In short, our result clearly demonstrates a versatile strategy to graft aldehyde groups on sensitive biopolymers under mild conditions that could be applied for various bioconjugation and biomedical applications such as drug delivery and regenerative medicine.

  • 45.
    Yan, Hongji
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Casalini, Tommaso
    Hulsart Billström, Gry
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Wang, Shujiang
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Oommen, Oommen P.
    Salvalaglio, Matteo
    Larsson, Sune
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Hilborn, Jöns
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Synthetic design of growth factor sequestering extracellular matrix mimetic hydrogel for promoting in vivo bone formation2018In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 161, p. 190-202Article in journal (Refereed)
    Abstract [en]

    Synthetic scaffolds that possess an intrinsic capability to protect and sequester sensitive growth factors is a primary requisite for developing successful tissue engineering strategies. Growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) is highly susceptible to premature degradation and to provide a meaningful clinical outcome require high doses that can cause serious side effects. We discovered a unique strategy to stabilize and sequester rhBMP-2 by enhancing its molecular interactions with hyaluronic acid (HA), an extracellular matrix (ECM) component. We found that by tuning the initial protonation state of carboxylic acid residues of HA in a covalently crosslinked hydrogel modulate BMP-2 release at physiological pH by minimizing the electrostatic repulsion and maximizing the Van der Waals interactions. At neutral pH, BMP-2 release is primarily governed by Fickian diffusion, whereas at acidic pH both diffusion and electrostatic interactions between HA and BMP-2 become important as confirmed by molecular dynamics simulations. Our results were also validated in an in vivo rat ectopic model with rhBMP-2 loaded hydrogels, which demonstrated superior bone formation with acidic hydrogel as compared to the neutral counterpart. We believe this study provides new insight on growth factor stabilization and highlights the therapeutic potential of engineered matrices for rhBMP-2 delivery and may help to curtail the adverse side effects associated with the high dose of the growth factor.

  • 46.
    Yan, Hongji
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Podiyan, Oommen
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Yu, Di
    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.
    Hilborn, Jöns
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Qian, Hong
    Varghese, Oommen P.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Chondroitin Sulfate-Coated DNA-Nanoplexes Enhance Transfection Efficiency by Controlling Plasmid Release from Endosomes: A New Insight into Modulating Nonviral Gene Transfection2015In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 25, no 25, p. 3907-3915Article in journal (Refereed)
    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.

  • 47. Yang, Hsiao-yin
    et al.
    Vonk, Lucienne A.
    Licht, Ruud
    van Boxtel, Antonetta M. G.
    Bekkers, Joris E. J.
    Kragten, Angela H. M.
    Hein, San
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Howard, Kenneth A.
    Oner, F. Cumhur
    Dhert, Wouter J. A.
    Creemers, Laura B.
    Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells2014In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 53, p. 35-44Article in journal (Refereed)
    Abstract [en]

    The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous cells. In conclusion, non-specific effects should not be overlooked in the application of RNAi for mesenchymal cell transfection and may need to be overcome for its effective therapeutic application.

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