Insights into siRNA transfection in suspension: Efficient gene silencing

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Insights into siRNA transfection in suspension: Efficient gene silencing in human mesenchymal stem cells encapsulated in hyaluronic acid hydrogel Maruthibabu Paidikondala, Ganesh N. Nawale, and Oommen P Varghese Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01712 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Insights into siRNA transfection in suspension: Efficient gene silencing in human mesenchymal stem cells encapsulated in hyaluronic acid hydrogel Maruthibabu Paidikondala,†,‡ Ganesh N. Nawale,†,‡ and Oommen P. Varghese*,† †

Translational Chemical Biology Laboratory, Polymer Chemistry Division, Department of Chemistry- Ångström Laboratory, Uppsala University, 751 21, Uppsala, Sweden ABSTRACT: Small interfering RNAs (siRNAs) are powerful tools for post-transcriptional gene silencing which offers enormous opportunities for tissue engineering applications. However, poor serum stability, inefficient intracellular delivery, and inevitable toxicity of transfection reagents are the key barriers for their clinical translation. Thus innovative strategies that allow safe and efficient intracellular delivery of the nucleic acid drugs at the desired site is urgently needed for a smooth clinical translation of therapeutically appealing siRNA-based technology. In this regard, we have developed an innovative siRNA transfection protocol that employs a short incubation time of just 5-minutes. This allows easy transfection in suspension followed by transplantation of the cells in a hyaluronic acid (HA) hydrogel system. We also report here the unique ability of siRNA to bind HA that was quantified by siRNA release and rheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA in suspension transfection conditions within 30-minutes using native HA, although removal of excess HA by centrifugation seem to be essential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈ 90 % gene silencing (with or without removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈ 40 % gene silencing after removal of excess reagent after 30 minutes. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improved knockdown (≈ 90 % and ≈ 60 % with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) as determined by PrestoBlueTM assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the protein levels by Western blot analysis. We believe this novel transfection method could be applied for in vivo applications as it allows minimal manipulation of cells that are to be transplanted and reduce toxicity. KEYWORDS: small interfering RNA (siRNA), transfection, hyaluronic acid (HA), hydrogel, gene regulation, human mesenchymal stem cells (hMSCs)

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1. INTRODUCTION Target specific gene silencing at post-transcription level employing siRNA has revolutionized modern medicine with a focus on precision medicine.1 The major advantage of such oligonucleotide drugs is their ability to target any mRNA of interest in a sequence-specific manner as it simply follows the Watson-Crick base pairing rules using RNA interference (RNAi) technology.1,

2

This allows silencing of protein

expression that cannot be reached by traditional methods such as monoclonal antibodies.3 Though this is the very promising, the cellular delivery of such RNAi drugs has limited its scope as compared to the small molecule drugs. This is because of the fact that such large negatively charged molecules do not penetrate the plasma membrane and also has very limited stability in the biological milieu.4 Thus, one of the focuses of research had been to develop transfection reagents that could deliver such synthetic molecules to cells under in vitro cell culture conditions. These reagents are usually composed of cationic polymers or lipids that bind siRNA by electrostatic interactions with the phosphate backbone. Such cationic reagents also impose cytotoxicity, which has limited its potential for clinical applications.5 Latest developments in the field aim to develop delivery carriers that are non-cationic at physiological pH (e.g. ionizable lipids) but acquire a cationic nature upon acidification within the intracellular compartment.6 Such a strategy reduces systemic toxicity and promotes cytosolic delivery of the cargo molecules by overcoming the endosomal barrier. We have recently shown that the coating of cationic DNA nanoparticles with anionic polymers such as chondroitin sulfate significantly improves gene delivery while minimizing toxicity.7 Although in vitro delivery of RNAi drugs had been successful, there has been a limited success in delivering these molecules to the target tissue in an in vivo setting. The FDA approval of the first RNAi drug Patisiran (for the treatment of hereditary transthyretin amyloidosis) has generated new hopes and excitement in the field.8 This drug employs an ionizable lipid with a cholesterol moiety that improves the pharmacokinetic and pharmacodynamic properties of the drug. The molecular conjugate of siRNA with a liver targeting ligand N-Acetyl-D-galactosamine (GalNAc) is the other most promising drug candidate that is currently tested in a clinical trial.9 Development of other siRNA-targeting ligand conjugates (e.g., antibody-siRNA)


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demonstrated that only a small subset of drug conjugate showed functional siRNA internalization.10 The challenges of in vivo gene silencing could be addressed for a local application if it is possible to perform ex vivo manipulation of cells followed by transplantation. Modification of primary cells for therapeutic applications is routinely performed for cell-based therapies, but it requires facilities for GMP scale production without any risk for batch-to-batch variation. We envisioned that if it would be possible to perform transient transfection experiment under suspension condition within a short time, it could significantly improve the current hurdles for ex vivo manipulation of cells. Traditionally suspension transfection involves lipoplex (cationic lipid-RNA complex) preparation followed by incubation of such lipoplexes with suspension cells for 24 to 72 h.11 We envisioned that transfection could indeed be possible in just 5–10 minutes, as cationic lipids have been reported to translocate the cargo molecules within the first 10 minutes by membrane fusion mechanism.12 To test our hypothesis, we selected primary bone marrow-derived mesenchymal stromal/stem cells as the model hard to transfect cells13 and an osteoinductive siRNA against Pleckstrin homology domaincontaining family O member 1 (PLEKHO1) also known as Casein kinase 2 interacting protein 1 (CKIP-1) as a model osteoinductive drug molecule.14 To validate our transfection strategy in a 3D system, we employed an HA-based hydrazonecrosslinked hydrogel (Scheme 1). Since carbohydrates are known to stabilize nucleic acids by CH-π interaction,15 we also investigated if such HA-based gel could act as a traditional gene-activated matrix that promotes stabilization of nucleic acids and facilitates cellular delivery.



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Scheme 1. Schematic representation of transfection of hMSCs in suspension with LipofectamineTM RNAiMAX or native HA and consequent encapsulation into HA hydrogel.

2. EXPERIMENTAL SECTION 2.1 Materials and reagents. Hyaluronic acid (HA, 200 kDa) was purchased from Lifecore Biomedical, LLC (Chaska, MN). Polyvinyl alcohol (PVA, 10 kDa, 89 % hydrolyzed) was purchased from Sigma-Aldrich, Sweden. Unmodified phosphoramidites (N6-benzoyl-rA, N2isobutyl-rG, N4-acetyl-rC, rU and dT) and solid supports were purchased from ChemGene Corporation (USA). Deblocking [3 % trichloroacetic acid in DCM (w/v)], activator [0.25M 5(ethylthio)-1H-tetrazole in acetonitrile], Cap A [2,6-lutidine/acetic anhydride/THF: 8/1/1 (v/v/v)],

Cap

B

[N-methylimidazole/pyridine/THF:

8/1/1

(v/v/v)]

and

oxidizer

[pyridine/iodine/water/THF: 90.54/9.05/0.41/0.43 (v/v/v/w)] reagents used for RNAs synthesis were

purchased

from

Sigma-Aldrich.

Lambda

35

UV/Vis

spectrophotometer

from

PerkinElmer instruments was used for spectroscopic analysis. Rheological properties of hydrogels were analyzed using Discovery Hybrid Rheometer (HR-2, TA Instruments) with custom-made parallel plate aluminium geometry of 8 mm diameter. 2.2

siRNA

synthesis.

Anti-PLEKHO1

CCUGAGUGACUAUGAGAAGdTdT,

siRNA

sequence

Antisense

(Sense strand:

strand:

5′5′-

CUUCUCAUAGUCACUCAGGdTdT) were synthesized using an automated solid-phase synthesizer (H8, K&A Synthesizer) with 2′-O-TBDMS protected A, C, G and U monomers with standard synthesis cycle. Cleavage from support and deprotection of base protecting groups were carried out by treating beads with AMA solution (1 mL, 41 % methylamine in


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water and 30 % aq. NH3 (1:1 v/v)). The 2′-O-TBDMS groups were deprotected using Et3N·3HF in DMSO. RNA was purified by 20 % denaturing PAGE (7 M urea) and recovered with TEN buffer. RNA samples were desalted using Sep-Pak (WAT020515, Waters) column. Pure RNA pellet was dissolved in water, and concentration was measured at 260 nm in UV−VIS spectrophotometer. 2.3 Polymer synthesis HA-aldehyde was prepared following our reported method.16 PVA-hydrazide was prepared following our reported method.17 2.4 Hydrogel preparation and release of siRNA from the hydrogel. HA-aldehyde derivative (8 mg, 10 % modification, 200 kDa) was dissolved in sterile PBS (500 µL, pH 7.4) and filtered through sterile 0.8 µm syringe filter (Pall Corp., East Hills, NY). PVA-hydrazide derivative (1.5 mg, 20 % modification, 10 kDa, 89 % hydrolyzed) was also dissolved in sterile PBS (500 µL, pH 7.4) and filtered through sterile 0.8 µm syringe filter (Pall Corp., East Hills, NY). Before the gelation, siRNA was incubated (static) for 10 minutes at room temperature with HA or PVA derivative. Both the polymer (120 µL of HA-siRNA and 80 µL of PVA or 120 µL of HA and 80 µL of PVA-siRNA) were mixed in a 24 well cell culture plate to make 200 µL total volume hydrogel. The mixture was allowed to incubate at 37 oC for 1 h in an incubator. Further 500 µL of sterile PBS (pH 7.4) was added to wells, and an aliquot of 3 µL was collected at different time points. Amount of RNA released in the PBS was calculated based on UV absorbance for siRNA at 260 nM. 2.5 Rheological characterization of HA hydrogel. HA-aldehyde and PVA-hydrazide solutions were prepared as described above. Both the solutions were filtered through 0.8 µm syringe filter (Pall Corp.). Before mixing the solutions for crosslinking, siRNA and RNAiMAX were added to HA solution for complexation. The two solutions were mixed by pipetting for 6–7 times at room temperature. The mixture was transferred to cylinder mold immediately after mixing. Molds were carefully covered with Parafilm and kept for 24 h at room temperature to obtain complete crosslinking. For determining the storage modulus (G′), 200 µL hydrogel was prepared and transferred to the rheometer plate. An oscillatory frequency sweep was performed with a controlled gap distance of 2000 µm at 1 Hz frequency and a strain rate of 1 %. 2.6 Cell Culture. Human bone marrow-derived Mesenchymal Stem Cells (hMSCs) were obtained from five different donors that were mixed and cultured together as described earlier18 in complete αMEM medium (Gibco) with 10 % hMSC grade fetal bovine serum (FBS; Cat# 12662029; Gibco), 1 % penicillin and streptomycin (Pen-Strep DE17-602E, Lonza) and 5 ng/ml recombinant human fibroblast



growth factor (FGF-2) (Cat# 100-18B; PEPROTECH). Cells were incubated at 37 °C and 5 % CO2 and the medium was refreshed every alternate day. 2.7 Adherent, suspension, and suspension followed by centrifugation transfection in 2D For adherent condition, one day before the experiment 35,000 cells per well were plated in a 24-well cell culture plate. RNAiMAX-based transfection was performed using the protocol from Thermofisher with 200 nM of PLEKHO1 siRNA per well and incubated at 37 °C with 5 % CO2 for 48 h or 72 h. For suspension transfection condition, we added RNAiMAX and siRNA complex in optiMEM medium to the tube containing 35,000 cells in cell culture medium having 10 % heatinactivated FBS and incubated for 10 minutes and further transferred to the respective wells and incubated at 37 °C with 5 % CO2 for 48 h or 72 h. For suspension followed by centrifugation conditions, we added RNAiMAX and siRNA complex in opti-MEM medium to the tube containing 35,000 cells in cell culture medium with 10 % heat-inactivated FBS. Thereafter, cells were briefly centrifuged for 5-minutes at 2150xg RCF, and the supernatant was aspirated. The pelleted cells were resuspended in 500 µL cell culture medium and transferred to the respective wells and incubated at 37 °C with 5 % CO2 for 48 h or 72 h. For HA-based siRNA transfection, the experiment was performed as follows. Briefly, HA (200 kDa) was dissolved in cell culture grade sterile PBS (4 mg/mL), to prepare the stock solution. Thereafter, 3.2 µL of the HA solution was mixed with 200 nM siRNA in a sterile Eppendrof tube, gently vortexed and incubated at room temperature for 5 minutes. Further experiments were performed as mentioned above. All experiments were done in triplicates and repeated twice. 2.8 Direct addition, suspension, suspension followed by centrifugation transfections in 3D HA-hydrogel with 1.9 % (w/v) of solid content was made as described earlier with ≈ 10 % aldehyde modified HA (HA-aldehyde)16 and ≈ 20 % hydrazide modified polyvinyl alcohol (PVA-hydrazide).17 Briefly, 16 mg/mL of aldehyde-modified HA was dissolved in sterile PBS while 3 mg/mL of PVAhydrazide was dissolved in DMEM medium and filtered with Acrodisc® Syringe Filter 0.8 µm Supor® Membrane (sterile) low protein binding Non-Pyrogenic (PN 4608) from PALL Life Sciences. Separately, cells were prepared from 2D culture through trypsinization. Later, 2.5 million cells per mL of PVA-hydrazide solution were prepared. To make a 200 µL HA-PVA hydrogel, 120 µL of HA-aldehyde solution was mixed with 80 µL of PVA-hydrazide solution with 200,000 cells in cell culture medium. For direct addition, HA hydrogels were prepared by adding 200 nM PLEKHO1 siRNA to 120 µL HA-aldehyde solution and mixed with PVA-hydrazide solution with 200,000 cells. The hydrogel was transferred to the 24-well cell culture plate, followed by 1 h incubation at 37 °C


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and 5 % CO2. Afterwards, 500 µL of the complete DMEM medium was added to hydrogels and incubated at 37 °C for 48 h or 72 h. For suspension transfection condition with RNAiMAX in HA-hydrogels, PLEKHO1 siRNA lipoplexes were prepared as stated above for 2D culture, which was added to 120 µL HAaldehyde solution in PBS. To this solution, 80 µL of PVA-hydrazide containing 200,000 cells was added and transferred to the 24-well cell culture plate. For suspension followed by centrifugation condition, we performed suspension transfection as described earlier. Thereafter, the cells were centrifuged at 2290xg RCF for 5 minutes; the supernatant was discarded to remove free reagent and the cells were collected as a pellet. The pelleted cells were suspended in a cell culture medium having PVA-hydrazide and mixed with the HAaldehyde solution to make hydrogel as described above. The hydrogel was transferred to the 24-well cell culture plate, followed by 1 h incubation at 37 °C and 5 % CO2. Afterwards, 500 µL of the complete DMEM medium was added to hydrogels and incubated at 37 °C for 48 h or 72 h. All experiments were performed in triplicates and repeated twice. 2.9 Cytotoxicity studies in 3D transfection. Different groups of HA hydrogels were prepared in triplicates as described above in black 96-well plate of a transparent bottom. After 3-days and 5-days post-transfection of hMSCs and consequent encapsulation into HA hydrogels, cytotoxicity was assessed using PrestoBlueTM assay kit and protocol from Invitrogen™ (Cat# A13261). Briefly, the medium was aspirated, and each well was given a 150 µl of 10 % PrestoBlueTM prepared in the cell culture medium and left to incubate for 1 h at 37 °C and 5 % CO2. Thereafter, fluorescence was measured using fluorescence spectrophotometer and the emission at 590 nm was determined with an excitation of 560 nm. The background was subtracted from all the obtained values, and average values were plotted from two independent experiments. All experiments were done in triplicates and repeated twice to determine the standard deviation. 2.10 RNA isolation and qRT-PCR. miRCURYTM RNA Isolation Kit (#300110) and protocol from EXIQON were used for RNA extraction. Briefly, medium from the culture plates was aspirated, and ice cold-PBS was employed for washing of cells twice. Afterwards, direct 350 µL of Lysis Solution was added to each well of the 24-well cell culture plate. The culture plate was gently tapped, and lysis buffer swirled around the well surface for 5 minutes to lyse the cells, and the resulting lysate was collected to a sterile Eppendorf tube. To this lysate containing Eppendorf tube, 200 µL of 96-100 % ethanol was added and vortexed for 10 seconds. Thereafter, the lysate was transferred to the RNA extraction column, followed by centrifugation at 20066xg RCF for 1 minute. Thereafter, we have performed column washing with the help of washing solution and consequent centrifugation at 20066xg RCF for 1 minute. The steps of column washing were repeated for two more times and the flow through was discarded. Into a fresh 1.7 mL elution tube, the column was properly placed and 30



µL of the elution buffer was added to the column, and then centrifugation was performed for 2 minutes at 286xg RCF and 1 minute at 20066xg RCF to collect the RNA. To extract RNA from HA hydrogels (200 µL each), the medium was aspirated, each gel was washed twice with ice-cold PBS, and they were collected into 2 mL sterile and low protein binding Eppendorf tubes. Thereafter, each Eppendorf tube containing gel was given 350 µL of the RNA lysis buffer along with 200 µL 96-100 % ethanol at 4 °C, followed by tissue lyser-assisted gel crushing and centrifugation at 20066xg RCF for 10 minutes at 4 °C. Thereafter, the supernatant was carefully collected to an RNA extraction column and followed further steps as explained above to extract RNA in 50 µL of the elution buffer. Subsequently, High Capacity RNA to cDNA kit and protocol from Applied Biosystems was used for the preparation of cDNA and qRT-PCR was performed with 500 ng cDNA with TaqMan® Fast Universal PCR Master Mix (2X), no AmpErase® UNG (Applied Biosystems) on MyiQTM Single color Real-Time PCR detection system from Bio-Rad. PLEKHO1 primers (Hs01062780_m1), ACTB (Hs01060665_g1) were obtained from Applied Biosystems (Cat # 4331182). Data from samples with a Ct (Cycle threshold) value less than or equals to 35 were considered for further analysis. All Ct values were regularized to internal control or house-keeping gene′s Ct value and differences in cycle number thresholds were obtained by utilizing comparative quantitation 2−ΔΔCT (ΔΔCT) technique to investigate siRNA induced gene silencing as explained previously.19 Concisely, calculations used to evaluate gene knockdown were as follows: ΔCT was measured as the mean cycle threshold for the target gene minus mean cycle thresholds for the house-keeping gene control, ACTB, each performed in triplicates: ΔCT = CT (target gene) – CT (endogenous control). Later, the ΔΔCT was calculated as the ΔCT of the target value minus the ΔCT of scrambled control (scr): ΔΔCT = ΔCT (target) – ΔCT (scr). Ultimately, the proportion of knockdown of the target gene was estimated as follows: Fold change = 2−ΔΔCT, then the percentage of Knockdown: = 100 * (1-fold change). 2.11 Western blotting. After 48 hrs of transfection of cells in 2D, the medium was removed, and the washing step was repeated twice with ice-cold PBS. Scraping of cells was performed, and cells were collected at 2290xg RCF for 5 minutes. Subsequently, 50 µL of the RIPA lysis buffer (Cat #R0278) with 1 % protease inhibitor cocktail from Sigma (Cat #P8340) was given to each cell pellet. Pipetting up and down of the cell pellets were carried out until they were dissolved in the lysis solution, briefly vortexed, incubated on ice for 30 minutes and centrifuged at 20066xg RCF at 4 °C for 10 minutes. The supernatant was collected without disturbing the pellet. To extract protein from HA hydrogels (200 µL each), the medium was aspirated, each gel was washed twice with ice-cold PBS, and they were collected into 2 mL sterile and low protein binding Eppendorf tubes. Thereafter, each Eppendorf tube containing gel was given 100 µL of the RIPA lysis buffer (Cat #R0278) with 2 % protease inhibitor cocktail from Sigma (Cat #P8340) at 4 °C, and did quick freezing in liquid nitrogen, followed


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by gel crushing using tissue lyser. Thereafter, we have performed centrifugation at 20066xg RCF at 4 °C for 20 minutes, and carefully collected supernatant. Protein concentrations were calculated using the Bradford method. Thereafter, SDS-PAGE was employed to separate 20 µg of soluble protein and then was transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). Primary antibodies raised in rabbit against CKIP-1 (Cat# sc-376355; Santa Cruz Biotechnology; 1:100 dilutions), and TBP (Cat# 44059; Cell Signaling Technology; 1:1000 dilutions) were used to probe protein bands. Anti-rabbit HRP-conjugated secondary antibodies (Cat# 170-6515; Bio-Rad; 1:2000 dilutions) were used for the detection of primary antibodies, followed by the target protein visualization with EMD Millipore ImmobilonTM Western Chemiluminescent HRP Substrate (ECL). Images were attained using ChemidocTM XRS + Systems from Bio-Rad.

3. RESULTS AND DISCUSSION Recently, several polymers-based gene-activated matrices have been developed for local delivery of siRNA in vivo, which includes hydrogels composed of cationic polymers such as chitosan, calcium crosslinked alginate, and atelocollagen based materials.20-22 Such materials stabilize nucleic acids by electrostatic interactions, similar to conventional cationic polymers. Though such materials have shown favorable in vitro and in vivo transfection efficiency, there are several challenges to overcome, which includes relatively low mechanical properties of the materials, nontoxic degradation of hydrogel components, the release of siRNA, recruitment of therapeutic cells to be transfected within the scaffold, and the therapeutic dose of siRNA.20 Thus biomaterials that possess the ability to enhance the transfection efficiency and controlled release of siRNA is highly desired.23 To develop such gene activated matrices that can sequester and stabilize nucleic acids we developed hydrazone crosslinked hyaluronic acid (HA) hydrogel. HA-based materials are very interesting for cell-based therapies as they are the only non-sulfated glycosaminoglycans within the extracellular matrix (ECM) that regulate a variety of cellular processes. This is because of its unique ability to sequester cell-secreted biologically active molecules such as cytokines and form the stem cell niche.24 We have recently reported that HA-hydrogels could be used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) in vivo to induce bone formation.25 To prepare HA-hydrogel, we synthesized the aldehyde-modified HA and hydrazidemodified polyvinyl alcohol (PVA) (Figure 1a). HA-aldehyde was synthesized as described earlier with ≈ 10 % modification,16 while the PVA-hydrazide derivative was



prepared following our previously described method with ≈ 20 % modification.17 HA gels were prepared at neutral pH and were loaded with free-siRNA to observe the release kinetics of nucleic acids upon gelation conditions. The release of siRNA from the hydrogel scaffold was quantitatively assessed using UV spectroscopy (Figure 1b). Interestingly, when siRNA was pre-incubated with HA-aldehyde polymer, a stable and slow release of siRNA was observed. Noticeably, only 60 % of siRNA was released from hydrogel even after 72 h of incubation (Figure 1b). However, when siRNA was pre-incubated with the PVA derivative nearly 80 % of RNA was released in 72 h (Figure 1b). This indicates that there is an interaction between HA and siRNA which regulates its release from the HA hydrogel. The observed controlled release of siRNA upon addition of HA prompted us to evaluate the rheological properties of the hydrogel upon addition of siRNA. As a control, we used siRNA free hydrogel and siRNA-RNAiMAX lipoplexes within the hydrogel network. To understand the structure-property relationship because of hydrogel crosslinking chemistry in the presence of RNA and RNAiMAX, we examined the storage modulus of the gels after 24 h to ensure complete curing of the materials. Interestingly, these experiments corroborated with the siRNA release experiments as we observed an increase in rigidity (≈ 150 Pa) of the gel as compared to the control gel without RNA (Figure 1c). This indicates that free siRNA acts as a crosslinker and increase the storage modulus of the hydrogel. In the case of RNAiMAX, we observed a marginal drop in storage modulus by ≈ 50 Pa, which is presumably because of an association of anionic HA with cationic lipids that prevent efficient mixing of the gel components. Taken together, our experiments revealed that there is indeed an association between native HA and siRNA presumably by hydrogen bonding and CH-π interactions, as observed with other carbohydrates.26, 27


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Figure 1. Hydrogel preparation and HA-siRNA interactions. (a) Scheme for HA and PVA hydrazone cross-linked hydrogel. (b) Graph showing the variance in the siRNA release profile from HA-PVA cross-linked hydrogel at neutral pH. Prior to HA-PVA cross-linked hydrogel, siRNA was incubated with either of the composite materials HA or PVA. (c) HA-PVA hydrogel rheological characterization. Storage modulus G′ as a function of frequency.

We further investigated if the structural characteristics could also reflect in functional gene knockdown in clinically relevant cells. We performed transfection experiments in hMSCs and targeted PLEKHO1, a gene widely observed in elderly patients that is considered as a key causative of osteoporosis.28 Proteins such as PLEKHO1 are also known to inhibit osteoblast differentiation by negatively regulating the canonical BMP-signaling pathway, which is essential for bone formation.29 Silencing of PLEKHO1 in vivo using cationic lipid-based carrier acted as an anabolic drug that resulted in augmented bone growth in an osteoporotic rat model.14 We, therefore, decided to deliver anti-PLEKHO1 siRNA to hMSCs in adherent as well as suspension

transfection

conditions

using RNAiMAX

and

HA.

Surprisingly,

quantitative RT-PCR experiments indicated that both RNAiMAX and HA successfully delivered functional siRNA to the adherent hMSCs that resulted in ≈ 90 % and ≈ 60 % knockdown (KD) of the target gene respectively (Figure 2a).



Figure 2. In vitro analysis of PLEKHO1 silencing through anti-PLEKHO1 siRNA transfection in hMSCs in 2D cell culture. (a) Quantitative RT-PCR results indicating the percentage knockdown (% KD) levels of PLEKHO1 mRNA in hMSCs after 48 h of transfection. Mean values ± SD of 2 independent experiments done in triplicates (n = 6) are presented. The Students t-test was used to determine statistical differences between pairs of groups (*** = P < 0.001; * = P < 0.05). (b) Western blot analysis is indicating PLEKHO1 protein levels after 72 h of transfection. The abbreviation scr = scrambled siRNA transfected cells; adh-HA/Lipo = HA/ RNAiMAX assisted transfection with adherent cells; susp-HA-Lipo = HA-RNAiMAX assisted transfection of cells in suspension; susp+centri-HA-Lipo = HA-RNAiMAX assisted transfection of cells in suspension followed by centrifugation. TATA-binding protein (TBP) was used as internal control.

On the other hand, suspension transfection using RNAiMAX resulted in ≈ 90 % knockdown within 5 minutes of incubation, however we did not observe any significant knockdown (≈ 10 %) when HA-siRNA was incubated with hMSCs for up to 1 h. We suspected this observation could be due to the blocking of the HA receptor CD44 due to excess reagent within the cell culture medium.30 We, therefore, repeated


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suspension transfection with HA and removed free transfection reagent by centrifugation after 30 min and 60 min of incubation. As anticipated such a strategy resulted in a modest ≈ 40 % knockdown of PLEKHO1 mRNA after 48 h (Figure 2a). We further evaluated the effect of mRNA knockdown at the protein levels by western blot analysis after 72 h of transfection. These experiments indicated a complete knockdown of the PLEKHO1 protein in case of adherent, suspension and suspension transfection followed by centrifugation method when RNAiMAX was used as the transfection agent. Interestingly, HA-based transfection also demonstrated complete knockdown of the target protein under adherent condition but not under suspension condition. The suspension transfection with HA could be partially rescued by removing the free reagent by centrifugation that resulted in a significant protein knockdown when compared to the scrambled control, corroborating with the qPCR results (Figure 2b). Our results obtained from 2D experiments prompted us to perform transfection experiment under suspension condition followed by encapsulation of the transfected stem cells in an HA hydrogel. Such an ex vivo manipulation of cells could be directly applied in a clinical setting using patient-derived bone marrow aspirate followed by transplantation at a defect site. Repetition of suspension transfection using RNAiMAX followed by encapsulation in a 3D gel indicated more than ≈ 80 % gene silencing while removing of the excess of the reagent by centrifugation after 5 minutes resulted in ≈ 90 % knockdown as quantified by qPCR analysis (Figure 3a). Interestingly, HA-based transfection also followed a similar trend as the 2D experiment with suspension followed by centrifugation yielding ≈ 60 % knockdown of PLEKHO1 (Figure 3a). In fact, suspension transfection with HA was more favorable in 3D cell culture (≈ 60 %) as compared to the 2D system (≈ 40 %). Of note, transfection experiments with HA did not show any significant knockdown when excess reagent was not removed indicating a competition of free HA with the HA-siRNA complex. Nevertheless, the HA-based transfection is unique and represents the first non-cationic transfection method that could be applied in a 3D system. The gene silencing efficiency was further evaluated at a protein level by western blot analysis, which corroborated with the qPCR results. These experiments indicated that both suspension transfection, as well as suspension followed by centrifugation after 5 minutes of incubation, resulting in complete knockdown of the target protein after 72 h when RNAiMAX was used as the transfection agent (Figure 3b). On the other hand, HA-



based suspension transfection demonstrated ≈ 50 % protein knockdown when excess reagent was removed after 30 minutes of incubation (Figure 3b).

Figure 3. In vitro analysis of PLEKHO1 gene silencing as a result of anti-PLEKHO1 siRNA transfection in hMSCs followed by encapsulation in HA hydrogel. (a) Quantitative RT-PCR results indicating the percentage knockdown (% KD) levels of PLEKHO1 mRNA in hMSCs after 48 h of transfection. Mean values ± SD of 2 independent experiments done in triplicates (n = 6) are presented. The Students t-test was used to determine statistical differences between pairs of groups (*** = P < 0.001; * = P < 0.05). (b) Western blot analysis is indicating PLEKHO1 protein levels after 72 h of transfection. The abbreviation scr = scrambled siRNA transfected cells; adh-HA-Lipo = HARNAiMAX assisted transfection with adherent cells; susp-HA-Lipo = HA-RNAiMAX assisted transfection of cells in suspension; susp+centri-HA-Lipo = HA-RNAiMAX assisted transfection of cells in suspension followed by centrifugation. TATA-binding protein (TBP) was used as internal control.

Since cellular toxicity is an important aspect that has limited the use of RNAi technology for in vivo application, we decided to evaluate the cytotoxicity of our transfection condition at 3and 5-day’s time periods using PrestoBlueTM assay (Figure 4). For this study, we performed


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suspension transfection experiments as discussed above (suspension and suspension followed by centrifugation) and encapsulated transfected cells inside the HA hydrogel. As a control, we loaded siRNA alone (without making lipoplexes) and lipoplexes into the HA hydrogel containing hMSCs. The cytotoxicity was measured with respect to the non-treated (NT) hMSCs in the hydrogel. These experiments showed that the RNAiMAX induced cytotoxicity to hMSCs with direct addition of lipoplexes to hydrogel being most toxic while suspension transfection followed by centrifugation being least toxic. These toxic effects were reduced over time, as the cytotoxicity after 5-days of experiment appears to be almost negligible as compared to that of the 3-days post-transfection experiment.

Figure 4. Cell viability studies of hMSCs transfected with anti- PLEKHO1 siRNA encapsulated in HA hydrogels after 3 and 5 days. The abbreviation NT = non-treated cells; siRNA = anti- PLEKHO1 siRNA alone; da-Lipo = direct addition of lipoplexes to MSC encapsulated in HA-hydrogel; s-Lipo = RNAiMAX assisted transfection of cells in suspension followed by encapsulation in HA-hydrogel; scLipo = RNAiMAX assisted transfection of cells in suspension followed by centrifugation and subsequent encapsulation in HA-hydrogel. Mean values ± SD of 3 independent experiments done in triplicates (n = 9) are presented. The Students t-test was used to determine statistical differences between pairs of groups (*** = P < 0.001; ** = P < 0.001* = P < 0.05).

4. CONCLUSIONS In summary, we demonstrate an efficient and non-toxic siRNA delivery method using conventional transfection reagent as well as native HA, which could be applied to



primary human MSCs. By performing the transfection experiments in suspension cells and for a short time of 5 minutes and 30 minutes for RNAiMAX and HA respectively, we offer a novel method for gene manipulation that can be applied directly to patientderived cells in a clinical setting. Our gene knockdown experiments in hMSCs successfully suppressed clinically relevant gene PLEKHO1 at the transcriptional and translational levels. HA-based intracellular delivery of siRNA not only provide new insight into the role of natural glycosaminoglycan in trafficking negatively charged nucleic acids but will also lead to the development of novel delivery carriers. The interaction of HA and siRNA was determined by siRNA release experiments and rheological characterization of siRNA encapsulated hydrogels. One of the major advantages of our transfection method in suspension is that it allows easy removal of excess reagent that improves transfection efficiency and increases biocompatibility and reduces cytotoxicity. Such an ex vivo transfection method addresses some of the major challenges in cellular manipulation for cell-based therapies and therefore could be translated for clinical application in a cost-effective manner. We believe our novel method of gene silencing will lead to new advancement in the field of gene-based therapies for clinical applications. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID ID: 0000-0001-8872-9928 Author Contributions ‡

These authors contributed equally to the paper.

Notes No competing financial interests have been declared. ACKNOWLEDGMENT We acknowledge financial support from Swedish Strategic Research ‘StemTherapy’ (Dnr 2009-1035), Swedish Foundation for Strategic Research (SSF, SBE13-0028) and ) and EU Framework Program-7 project FP7/2007-2013/607868 (Marie Curie Actions- iTERM). REFERENCES


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Insights into siRNA transfection in suspension: Efficient gene silencing

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