| 1. |
- Paidikondala, Maruthibabu, 1985-, et al.
(författare)
-
An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier : The First Biomimetic, Anionic, Non-Viral Transfection Method
- 2019
-
Ingår i: Angewandte Chemie International Edition. - : WILEY-V C H VERLAG GMBH. - 1433-7851 .- 1521-3773. ; 58:9, s. 2815-2819
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 2. |
|
|
| 3. |
- Paidikondala, Maruthibabu, 1985-, et al.
(författare)
-
Impact of Hydrogel Cross-Linking Chemistry on the in Vitro and in VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-2
- 2019
-
Ingår i: ACS Applied Bio Materials. - : American Chemical Society (ACS). - 2576-6422.
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 4. |
- Paidikondala, Maruthibabu, 1985-, et al.
(författare)
-
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
-
Ingår i: International Journal of Molecular Sciences. - Basel, Switzerland : MDPI. - 1661-6596 .- 1422-0067. ; 20:1
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 5. |
- Paidikondala, Maruthibabu, 1985-, et al.
(författare)
-
Insights into siRNA Transfection in Suspension : Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel
- 2019
-
Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 20:3, s. 1317-1324
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Paidikondala, Maruthibabu, 1985-
(författare)
-
Regulating Gene Expression to Promote Osteoblastic Differentiation of Stem Cells
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
