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Paidikondala, MaruthibabuORCID iD iconorcid.org/0000-0003-2896-2765
Publications (10 of 11) Show all publications
Paidikondala, M., Rangasami, V. K., Nawale, G. N., Casalini, T., Perale, G., Kadekar, S., . . . Varghese, O. P. (2019). An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method. Angewandte Chemie International Edition, 58(9), 2815-2819
Open this publication in new window or tab >>An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method
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2019 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 58, no 9, p. 2815-2819Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2019
Keywords
nanoparticles, extracellular matrix, hyaluronic acid, RNAi, transfection
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-379591 (URN)10.1002/anie.201900099 (DOI)000459807200045 ()30644615 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SBE13-0028
Available from: 2019-03-18 Created: 2019-03-18 Last updated: 2019-03-18Bibliographically approved
Paidikondala, M., Wang, S., Hilborn, J., Larsson, S. & Varghese, O. P. (2019). Impact of Hydrogel Cross-Linking Chemistry on the in Vitro and in VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-2. ACS Applied Bio Materials
Open this publication in new window or tab >>Impact of Hydrogel Cross-Linking Chemistry on the in Vitro and in VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-2
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2019 (English)In: ACS Applied Bio Materials, ISSN 2576-6422Article in journal, Editorial material (Refereed) Published
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.

Keywords
bone tissue engineering; cross-linking chemistry; drug delivery; hyaluronic acid; regenerative medicine
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-382321 (URN)10.1021/acsabm.9b00060 (DOI)
Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-04-24
Paidikondala, M., Kadekar, S. & Varghese, O. P. (2019). Innovative strategy for 3D transfection of primary human stem cells with BMP-2 expressing plasmid DNA: A clinically translatable strategy for ex vivogene therapy. International Journal of Molecular Sciences, 20(1), Article ID 56.
Open this publication in new window or tab >>Innovative strategy for 3D transfection of primary human stem cells with BMP-2 expressing plasmid DNA: A clinically translatable strategy for ex vivogene therapy
2019 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 1, article id 56Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Basel, Switzerland: MDPI, 2019
Keywords
hydrogel; DNA; transfection; ex vivo; hyaluronic acid
National Category
Biomaterials Science
Identifiers
urn:nbn:se:uu:diva-369655 (URN)10.3390/ijms20010056 (DOI)000459747700056 ()30583610 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, FP7/2007-2013/607868Swedish Foundation for Strategic Research , SBE13-0028
Available from: 2018-12-14 Created: 2018-12-14 Last updated: 2019-03-18Bibliographically approved
Paidikondala, M., Nawale, G. N. & Varghese, O. P. (2019). Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel. Biomacromolecules, 20(3), 1317-1324
Open this publication in new window or tab >>Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel
2019 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 20, no 3, p. 1317-1324Article in journal (Refereed) Published
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.

National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-379682 (URN)10.1021/acs.biomac.8b01712 (DOI)000461270500019 ()30642167 (PubMedID)
Funder
Swedish Foundation for Strategic Research , 2009-1035Swedish Foundation for Strategic Research , SBE13-0028EU, FP7, Seventh Framework Programme, FP7/2007-2013/607868
Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-04-12Bibliographically approved
Paidikondala, M. (2019). Regulating Gene Expression to Promote Osteoblastic Differentiation of Stem Cells. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Regulating Gene Expression to Promote Osteoblastic Differentiation of Stem Cells
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bone is a tissue that heals by itself, unless the defect is too large (critical size). Today, novel regenerative medicine approaches have emerged as an alternative to treat such defects. This thesis explores alternative therapeutic strategies for bone tissue engineering which are biocompatible and clinically translatable. Many types of scaffolds that can act as reservoirs for growth factors such as rh-BMP-2 have been developed for bone tissue engineering in the past. However, the role of cross-linking chemistries that are employed to make hydrogels on the integrity and function of the loaded growth factors is not well understood. In this thesis, we have explored the influence of cross-linking chemistry on rh-BMP-2 integrity and bioactivity both in-vitro and in-vivo. These studies have demonstrated that thiol-Michael addition cross-linking chemistry greatly affects the integrity and bio-functionality of the loaded protein BMP-2 and leads to poor bone formation in an in-vivo rat model. On the other hand, hydrogels employing hydrazone chemistry did not significantly affect the integrity and bioactivity of BMP-2, which lead to a superior bone formation in-vivo. Since the high dose of rh-BMP-2 is known to confer many side effects, alternative ex-vivo strategies involving transient transfection of BMP-2 expressing plasmid DNA and silencing of anti-osteogenic genes using siRNA are developed. Our optimized method involves rapid transfection of hMSCs in suspension (5 minutes) with plasmid DNA followed by centrifugation and encapsulation in a hydrogel not only reduced cytotoxicity but also lead to efficient osteoblast differentiation of stem cells. Furthermore, this thesis presents the role of ECM-derived polymer HA in interacting with siRNA and trafficking across the plasma membrane, presumably through CD44 receptors and successfully silencing the target gene in-vitro. We explored the potential of such a non-cationic transfection method to deliver functional siRNA (anti-Pleckho-1 siRNA) in MSCs and compared it with commercially available cationic lipid LipofectamineTMRNAiMAX, using our optimized suspension transfection method. Our novel ex-vivo strategy employing HA hydrogels enabled efficient silencing of BMP-2 signaling pathway antagonist Pleckho-1 while avoiding the cytotoxicity issues in 3D, which further qualifies them for potential clinical application for cell-based therapies. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 106
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1758
National Category
Polymer Chemistry
Research subject
Chemistry with specialization in Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-369660 (URN)978-91-513-0540-0 (ISBN)
Public defence
2019-02-13, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-01-23 Created: 2018-12-15 Last updated: 2019-02-18
Neddermeyer, A. H., Hultenby, K., Paidikondala, M., Schuchman, R. M. & Bidokhti, M. R. M. (2018). Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy. Current Therapeutic Research: Clinical and Experimental, 88, 8-17
Open this publication in new window or tab >>Investigating Tick-borne Flaviviral-like Particles as a Delivery System for Gene Therapy
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2018 (English)In: Current Therapeutic Research: Clinical and Experimental, ISSN 0011-393X, E-ISSN 1879-0313, Vol. 88, p. 8-17Article in journal (Refereed) Published
Abstract [en]

Background: Research on the biogenesis of tick-borne encephalitis virus (TBEV) would benefit gene therapy. Due to specific arrangements of genes along the TBEV genome, its viral-like particles (VLPs) could be exploited as shuttles to deliver their replicon, which carries therapeutic genes, to immune system cells.

Objective: To develop a flaviviral vector for gene delivery as a part of gene therapy research that can be expressed in secretable VLP suicidal shuttles and provide abundant unique molecular and structural data supporting this gene therapy concept.

Method: TBEV structural gene constructs of a Swedish Torö strain were cloned into plasmids driven by the promoters CAG and CMV and then transfected into various cell lines, including COS-1 and BHK-21. Time-course sampling of the cells, culture fluid, cell lysate supernatant, and pellet specimens were performed. Western blotting and electron microscopy analyses of collected specimens were used to investigate molecular and structural processing of TBEV structural proteins.

Results: Western blotting analysis showed differences between promoters in directing the gene expression of the VLPs constructs. The premature flaviviral polypeptides as well as mature VLPs could be traced. Using electron microscopy, the premature and mature VLP accumulation in cellular compartments—and also endoplasmic reticulum proliferation as a virus factory platform—were observed in addition to secreted VLPs.

Conclusions: The abundant virologic and cellular findings in this study show the natural processing and safety of inserting flaviviral structural genes into suicidal VLP shuttles. Thus, we propose that these VLPs are a suitable gene delivering system model in gene therapy.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE INC, 2018
Keywords
tick-borne encephalitis virus, flavivirus, viral-like particles, gene therapy, delivery system, electron microscopy, CAG promoter, CMV promoter
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-364517 (URN)10.1016/j.curtheres.2017.10.003 (DOI)000440577200002 ()
Funder
Knowledge Foundation
Available from: 2018-11-05 Created: 2018-11-05 Last updated: 2018-11-05Bibliographically approved
Cholleti, H., Paidikondala, M., Munir, M., Hakhverdyan, M. & Baule, C. (2013). Equine arteritis virus induced cell death is associated with activation of the intrinsic apoptotic signalling pathway. Virus Research, 171(1), 222-226
Open this publication in new window or tab >>Equine arteritis virus induced cell death is associated with activation of the intrinsic apoptotic signalling pathway
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2013 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 171, no 1, p. 222-226Article in journal (Refereed) Published
Abstract [en]

Equine arteritis virus (EAV) causes a respiratory and reproductive disease in horses, equine viral arteritis. Though cell death in infection with EAV is considered to occur by apoptosis, the underlying molecular mechanism has not been extensively elucidated. We investigated the expression of mRNA of pro-apoptotic and caspase genes during EAV infection in BHK21 cells, a well-established cell type for EAV replication. Using a SYBR Green real-time PCR, mRNA of p53, Bax, caspase 3 and caspase 9 were found up-regulated in a time dependent manner in EAV infected cells. Western blot analysis for caspase 3 and caspase 9 showed expression of cleaved forms of these proteins during EAV infection. In addition, a luminescence-based cell assay for caspase 3/7 activation as a hallmark in apoptosis confirmed apoptotic cell death. The findings demonstrate that cell death in EAV infected BHK21 cells results from apoptosis mediated through the intrinsic signalling pathway.

Keywords
3/7, Apoptosis, Bax, Caspase 9, Equine arteritis virus, P53, Real-time PCR
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-195151 (URN)10.1016/j.virusres.2012.10.004 (DOI)000315607300026 ()
Available from: 2013-02-22 Created: 2013-02-21 Last updated: 2017-12-06Bibliographically approved
Mottahedin, A., Paidikondala, M., Cholleti, H. & Baule, C. (2013). NF-kappa B activation by equine arteritis virus is MyD88 dependent and promotes viral replication. Archives of Virology, 158(3), 701-705
Open this publication in new window or tab >>NF-kappa B activation by equine arteritis virus is MyD88 dependent and promotes viral replication
2013 (English)In: Archives of Virology, ISSN 0304-8608, E-ISSN 1432-8798, Vol. 158, no 3, p. 701-705Article in journal (Refereed) Published
Abstract [en]

NF-kappa B, a family of transcription factors involved in different cell functions and immune responses is targeted by viruses. The mechanism of NF-kappa B signalling and its role in replication of EAV have not been investigated. We demonstrate that EAV infection in BHK-21 cells activates NF-kappa B, and this activation was found to be mediated through the MyD88 pathway. Infection of IKK beta(-/-) murine embryo fibroblasts (MEFs), which are deficient in NF-kappa B signalling, resulted in lower virus titre, less cytopathic effect, and reduced expression of viral proteins. These findings implicate the MyD88 pathway in EAV-induced NF-kappa B activation and suggest that NF-kappa B activation is essential for efficient replication of EAV.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-197630 (URN)10.1007/s00705-012-1515-4 (DOI)000315561900021 ()
Available from: 2013-04-03 Created: 2013-04-02 Last updated: 2017-12-06Bibliographically approved
Paidikondala, M., Rangasami, V. K., Nawale, G. N., Casalini, T., Perale, G., Podiyan, O. & Varghese, O. P.Hyaluronic acid facilitates non-cationic siRNA delivery and gene silencing in CD44 positive cells.
Open this publication in new window or tab >>Hyaluronic acid facilitates non-cationic siRNA delivery and gene silencing in CD44 positive cells
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(English)Manuscript (preprint) (Other academic)
National Category
Biomaterials Science
Identifiers
urn:nbn:se:uu:diva-369657 (URN)
Available from: 2018-12-14 Created: 2018-12-14 Last updated: 2018-12-15
Paidikondala, M., Nawale, G. N. & Varghese, O. P.New insight on siRNA transfection in three dimensions: Improved gene silencing in human mesenchymal stem cells encapsulated in hyaluronicacid hydrogel.
Open this publication in new window or tab >>New insight on siRNA transfection in three dimensions: Improved gene silencing in human mesenchymal stem cells encapsulated in hyaluronicacid hydrogel
(English)Manuscript (preprint) (Other academic)
National Category
Biomaterials Science
Identifiers
urn:nbn:se:uu:diva-369658 (URN)
Available from: 2018-12-14 Created: 2018-12-14 Last updated: 2018-12-15
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ORCID iD: ORCID iD iconorcid.org/0000-0003-2896-2765

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