Transforming Hairpin-like siRNA-Based Spherical Nucleic Acids into Biocompatible Constructs

Abstract

The design and synthesis of hairpin-like small interfering RNA spherical nucleic acids (siRNA-SNAs) based upon biocompatible liposome nanoparticle cores are described. The constructs have been characterized by gel electrophoresis, dynamic light scattering, and OliGreen-based oligonucleotide quantification. These siRNA-SNA nanoconstructs enter cells 20 times more efficiently than linear siRNA in as little as 4 h, while exhibiting a 4-fold reduction in cytotoxicity compared to conventional siRNA-SNAs composed of gold nanoparticle cores. Importantly, these siRNA-SNA constructs effectively inhibit angiogenesis in vitro by silencing vascular endothelial growth factor, a key mediator of angiogenesis in a multitude of diseases, in human umbilical vein endothelial cells. This work shows how hairpin architectures can be chemically incorporated into biocompatible SNAs in a way that retains advantageous SNA properties and maximizes gene regulation capabilities.

Graphical Abstract

INTRODUCTION

Small interfering RNA (siRNA) is a short, double-stranded RNA molecule that can, in principle, be designed to silence any target gene of interest.^1-3 siRNAs have tremendous potential as gene regulation therapeutics, but their widespread clinical use has been restricted by stability and delivery limitations.^4-6 This is because siRNAs are rapidly degraded in biological fluids,⁷ have unfavorable pharmacokinetics,^8,9 and cannot independently enter cells.^10,11 To overcome these limitations, siRNAs have been transformed into spherical nucleic acids (SNA) by radially arranging them around a nanoparticle core, imparting them with properties distinct from those of linear siRNA.^12,13 In particular, they are resistant to nuclease degradation compared to linear siRNAs, can independently and efficiently enter tissues and cells via scavenger receptor-mediated endocytosis, and typically exhibit longer circulating half-lives, thereby favoring delivery to target sites (e.g., tumors) when intravenously administered.^14,15

Conventional siRNA-SNAs consist of thiolated passenger strands attached to a gold nanoparticle core, with guide strands hybridized to the passenger strands.¹⁴ siRNA-SNAs using this architecture have shown therapeutic success in in vivo models of impaired wound healing¹⁶ and psoriasis¹⁷ and have progressed to a first-in-human clinical trial for glioblastoma.^15,18 However, this design has several limitations that restricts its clinical suitability. First, the hybridized attachment strategy severely limits the number of siRNA duplexes that can be loaded on each SNA. Due to the high charge density near the SNA core, electrostatic repulsion causes the majority of guide strands to dissociate, leaving few siRNA duplexes intact.¹⁹ The development of a hairpin-like siRNA attachment strategy that maximizes siRNA duplex loading has recently been reported, although this has only been demonstrated using a gold-core siRNA-SNA (siRNA-AuSNA).²⁰ The second limitation of the prototypical siRNA-SNA design is its gold nanoparticle core. Indeed, gold nanoparticles are not biodegradable, increase the cost and molecular weight of constructs, and are potentially cytotoxic at high concentrations.^19-21 An siRNA-SNA based upon a biocompatible nanoparticle core with a similar hairpin architecture could overcome many of these limitations.

SNAs developed using a liposome core have shown promise as drugs for the treatment of a wide variety of diseases, including certain forms of cancer and infectious disease.^22-28 Liposome cores are attractive alternatives to gold nanoparticles in siRNA-SNAs because they are inherently nontoxic and could increase siRNA bioavailability. Herein, we describe the synthesis and characterization of hairpin-like siRNA-based liposomal SNAs (siRNA-LSNAs) and establish this class of constructs as a potential next-generation siRNA-SNA therapeutic. In addition to the successful synthesis of the hairpin-like siRNA-LSNA, we show the following: 1) the hairpin-like architecture increases siRNA duplex loading on LSNAs by 3-fold in comparison to the hybridized architecture, 2) hairpin-like siRNA-LSNAs retain the SNA property of independently and efficiently entering cells, 3) the biocompatible liposome core reduces SNA cytotoxicity by 4-fold compared to a gold nanoparticle core, and 4) hairpin-like siRNA-LSNAs possess gene silencing capabilities, demonstrated by the targeted inhibition of vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVEC), thus exhibiting the gene regulation capabilities of this new construct.

RESULTS AND DISCUSSION

Characterization of Hairpin-like siRNA-LSNAs.

Hairpin-like siRNA-LSNAs were synthesized by adding hairpin-like siRNA with a hydrophobic anchor to a liposome core (Figures 1A and S1). Since the identity of the lipophilic moiety used to insert oligonucleotides into the liposomal membrane can impact LSNA stability,²⁹ a variety of hydrophobic anchors at the turn of the hairpin were investigated, including tocopherol, cholesterol, double tocopherol (two tocopherol phosphoramidites linked together), and double cholesterol (Figure S2). To evaluate LSNA formation, linear hairpin-like siRNAs and LSNA formulations were ran through an agarose gel and stained with SYBR Gold to indicate the location of the siRNA. However, only the single tocopherol anchor resulted in successful LSNA formation (Figure 1B). While free tocopherol hairpin-like siRNA ran to the bottom of the gel, the siRNA-LSNA showed a prominent mobility shift indicating successful LSNA formation,^22,30 with excess unattached siRNA running to the bottom of the gel. To confirm that this higher band was an LSNA consisting of siRNA attached to a liposome core and not a micelle composed of tocopherol hairpin-like siRNAs, an LSNA containing a rhodamine-labeled lipid was also compared. When analyzing the gel for rhodamine, a band indicating the liposome overlapped at the same location as the band indicating the siRNA, confirming that the siRNA and liposome are colocalized in the gel and are indeed an LSNA.

Figure 1. Hairpin-like siRNA-LSNA characterization.

(A) Chemical structure of hairpin-like siRNA-LSNA. (B) Agarose gel showing hairpin-like siRNA-LSNA formation. (C) DLS of bare liposomes and hairpin-like siRNA-LSNAs. Error bars represent the standard deviation of 10 measurements. (D) siRNA duplex loading on LSNAs using hybridized (hyb.) and hairpin-like (HP) architecture. Error bars represent the standard deviation of 3 technical replicates. Statistical analysis was performed using an unpaired t-test, where “****” represents a P value of <0.0001.

While the other anchors are more hydrophobic, which would hypothetically lead to more stable LSNAs, hairpin-like siRNAs with those anchors primarily formed micelles instead of LSNAs (example shown with double tocopherol: Figure S3). Dominant micelle formation is likely due to the strong amphiphilicity of these molecules, arising from the high hydrophobicity of the anchor and the strong hydrophilicity of the 44 RNA nucleotides. While previous findings have demonstrated that more hydrophobic anchors lead to more stable LSNAs, those experiments were done using single-stranded DNA that contained half as many nucleotides as the hairpin-like siRNA.²⁹ Our results demonstrate that if anchors are too hydrophobic and the nucleic acids are too large (i.e., too hydrophilic), prominent micelle formation can impede the formation of LSNAs. Thus, successful LSNA formation requires an anchor that is sufficiently hydrophobic to stably insert into liposomes but not so hydrophobic to the extent that micelle formation is dominant over LSNA formation. As such, liposome cores in this study were composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), since LSNAs with a DPPC core are more stable than LSNAs with cores composed of other lipids.³⁰ Following synthesis, LSNAs were washed with 1× Dulbecco’s phosphate-buffered saline (DPBS) using 100K molecular weight cutoff centrifugal filters to remove unattached siRNA. LSNA synthesis was further confirmed via dynamic light scattering (DLS) (Figures 1C and S4). As expected, a size increase was observed between bare liposomes and liposomes functionalized with hairpin-like siRNAs, indicating LSNA formation.

The amount of siRNA duplexes on the LSNA was quantified using an OliGreen assay (Figure 1D). Hairpin-like siRNA-LSNAs had an average of 29 siRNA duplexes per LSNA, with a surface loading density of 3.7 pmol/cm². The loading density of the hairpin-like siRNA-LSNAs was compared with that of LSNAs containing siRNAs attached via the prototypical tocopherol-anchored hybridized architecture (Figure S2). The hairpin-like architecture enables a significantly higher siRNA duplex loading than the hybridized architecture for siRNA-LSNAs (29 duplexes/particle vs. 11 duplexes/particle, respectively), similar to the increase observed with siRNA-AuSNAs.²⁰ The hybridized siRNA-LSNAs likely suffer from lower duplex loading due to the high negative charge density from the siRNA near the core causing the majority of guide strands to dissociate from the SNA.¹⁹ The hairpin-like architecture covalently attaches the passenger and guide strand to each other, preventing guide strand dissociation and enabling higher duplex loading on the SNA. This result shows that the ability of the hairpin-like architecture to increase siRNA duplex loading is not limited to siRNA-AuSNAs but can be applied to other cores as well.

Hairpin-Like siRNA-LSNAs Efficiently Enter Cells.

To our knowledge, this is the first example of an siRNA-SNA with a liposome core. Thus, it is necessary to confirm that the hairpin-like siRNA-LSNAs exhibit the important SNA property of independently and effectively entering cells. HUVECs were treated with Cy5-labeled linear siRNA and Cy5-labeled hairpin-like siRNA-LSNAs. Flow cytometry analysis showed that the LSNAs had accumulated in the cells much more efficiently than linear siRNA at both 4 h and 24 h timepoints (Figure 2A), with up to a 20-fold increase observed at 4 h. This finding was confirmed with confocal microscopy, which showed that LSNAs prominently accumulate in cells while linear siRNA had no observable entry (Figure 2B). Thus, hairpin-like siRNA-LSNAs retain the property of independently entering cells.

Figure 2. Cellular uptake of hairpin-like siRNA-LSNAs.

(A) Hairpin-like siRNA-LSNAs efficiently enter HUVECs in comparison to linear siRNAs, as measured by flow cytometry. Error bars represent the standard deviation of 3 biological replicates. (B) Confocal microscopy images of Cy5-labeled linear siRNAs and hairpin-like siRNA-LSNAs internalized by HUVECs (blue: DAPI stain indicating nucleus, red: Cy5 indicating siRNA), scale bar: 10 μm. Statistical analysis was performed using an unpaired t-test, where “****” represents a P value of <0.0001.

Previous siRNA-SNAs were designed using a gold nanoparticle core that may potentially be cytotoxic at high concentrations.^19,20 As a test of the biocompatibility of hairpin-like siRNA-LSNAs compared to hairpin-like siRNA-AuSNAs, HUVECs were treated with increasing concentrations of hairpin-like siRNA-SNAs containing either core for 48 h and assessed for cell viability (Figure 3A). The median lethal dose (LD₅₀) of hairpin-like siRNA-LSNAs was significantly higher (4-fold) than that of hairpin-like siRNA-AuSNAs (Figure 3B). The lower cytotoxicity of hairpin-like siRNA-LSNAs is an example of their improved biocompatibility compared to SNAs based on the prototypical gold nanoparticle core.

Figure 3. Cytotoxicity of hairpin-like siRNA-SNAs.

(A) Cytotoxicity of hairpin-like siRNA-LSNAs and hairpin-like siRNA-AuSNAs. Error bars represent the standard deviation of 3 biological replicates. (B) LD₅₀ of hairpin-like siRNA-LSNAs and hairpin-like siRNA-AuSNAs, derived from curves in (A). Error bars represent the standard deviation of 3 biological replicates. Statistical analysis was performed using an unpaired t-test, where “****” represents a P value of <0.0001.

Hairpin-like siRNA-LSNAs Demonstrate Silencing Activity in Vitro.

The hairpin-like architecture has been previously shown to have no detrimental effect on siRNA gene silencing activity.²⁰ In this study, the gene silencing activity of hairpin-like siRNA-LSNAs was investigated using a tube formation assay. Specifically, hairpin-like siRNA-LSNAs were designed to target VEGF, resulting in the inhibition of angiogenesis. Excessive blood vessel formation as a result of VEGF upregulation is a hallmark of many diseases, including cancers, making it an attractive target for gene silencing.^31-33 As an in vitro model of angiogenesis, HUVECs were incubated atop a Matrigel basement membrane matrix with the growth factors necessary to induce capillary blood vessel formation. Under these conditions, HUVECs form long branches of interconnected tubes that resemble a network of capillaries. HUVECs with reduced VEGF expression will not form tubes but instead remain as rounded cells.

HUVECs were treated with nontargeting or VEGF-targeting hairpin-like siRNA-LSNAs, then embedded in Matrigel. Tube formation was analyzed at 24 h and 48 h after treatment via light microscopy (Figure 4). Untreated HUVECs and those treated with nontargeting SNAs showed an extensive network of tubes at both time points, indicating prevalent angiogenesis. However, HUVECs treated with VEGF-targeting SNAs showed dramatically reduced tube formation, with nearly complete inhibition of angiogenesis at the 48 h time point, indicating successful VEGF silencing. The degree of tube formation was quantified via ImageJ’s Angiogenesis Analyzer tool, and it was found that treatment with VEGF-targeting SNAs reduced tube formation by four-fold (Figure S5). Thus, hairpin-like siRNA-LSNAs demonstrate sequence-specific silencing that results in an observable phenotypic change.

Figure 4. VEGF-targeting siRNA-LSNAs inhibit angiogenesis in vitro.

Optical microscope images of HUVECs grown in Matrigel basement membrane matrix following treatment with siRNA-LSNAs for 24 h and 48 h, scale bar: 50 μm. Yellow arrows indicate capillary-like tubes.

CONCLUSIONS

In summary, we describe the synthesis, characterization, and gene regulation capabilities of novel hairpin-like siRNA-based SNAs with biocompatible liposome cores. Hairpin-like siRNA-LSNAs are more biocompatible than prototypical gold-core siRNA-SNAs and exhibit efficient cellular uptake and effective gene silencing. In addition, the effects of SNA structure on function can be utilized to design and synthesize constructs that potentially are more therapeutically relevant. The combined structural features of these constructs, including the hairpin architecture, the hydrophobic tocopherol anchor, and the stable and biocompatible DPPC liposome core collectively lead to their composite properties, which include enhanced gene regulation capabilities. The proof-of-concept study with the VEGF-targeting siRNA-LSNAs illustrates the potential of these constructs for treating a wide variety of diseases where gene knockdown is a therapeutically relevant pathway. This includes many forms of cancer, where inhibiting angiogenesis is a viable strategy,^31,32,34-36 and diseases of the eye characterized by abnormal blood vessel formation.^33,37-39

EXPERIMENTAL SECTION

Oligonucleotide Synthesis.

RNA oligonucleotides were synthesized on a MerMade 12 system (BioAutomation) using 2’-O-triisopropylsilyloxymethyl-protected phosphoramidites (ChemGenes). Hairpin-like siRNA consisted of RNA base cyanoethyl phosphoramidites (the guide portion of the strand) (Glen Research), two spacer-18 (18-O-dimethyoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)] phosphoramidites, a hydrophobic anchor phosphoramidite (the hairpin turn and liposome attachment moiety), two spacer-18 phosphoramidites, and RNA base cyanoethyl phosphoramidites (the passenger portion of the strand). Tocopherol, cholesterol, double tocopherol (two tocopherol phosphoramidites linked together), and double cholesterol were investigated as the hydrophobic anchor, and a single tocopherol phosphoramidite was selected as the hydrophobic anchor for LSNA synthesis. For gold nanoparticle-core SNAs, the hairpin-like siRNA contained a dithiol serinol phosphoramidite in place of the hydrophobic anchor as previously reported.²⁰ The hybridized siRNA system consisted of separate passenger and guide strands. The passenger strand consisted of RNA base cyanoethyl phosphoramidites, two spacer-18 phosphoramidites, and a tocopherol phosphoramidite as the hydrophobic anchor. The guide strand consisted of RNA base cyanoethyl phosphoramidites. After synthesis, RNA oligonucleotides were deprotected following the manufacturer’s protocol (Bioautomation). Deprotected RNA oligonucleotides were purified using high-performance liquid chromatography on a Zorbax C4 column using 0.1 M triethylammonium acetate and acetonitrile as the solvents. The 5’-DMT group was removed from the purified RNA oligonucleotides via treatment with 20% acetic acid at room temperature for 2 h and extracting 3 times with ethyl acetate. The oligonucleotide solution was lyophilized and suspended in DNase/RNase-free water. The masses of the oligonucleotides were measured using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Oligonucleotides were mixed with 2’,4’-dihydroxyacetophenone (DHAP) and dried on a MALDI plate. MALDI-TOF was performed using an Autoflex III Smartbeam MALDI-TOF mass spectrometer. Oligonucleotide concentrations were determined using UV-vis spectroscopy. The oligonucleotide sequences used in this work are listed in Table S1.

LSNA Synthesis.

Thin films of DPPC were prepared by adding 400 μL of 25 mg/mL DPPC in chloroform (Avanti Polar Lipids) to a scintillation vial, evaporating the chloroform under a stream of nitrogen, lyophilizing, then storing under argon at −20 °C. To prepare liposomes, the thin film was rehydrated in 1 mL 1× DPBS, frozen in liquid nitrogen, and sonicated in a 45 °C water bath for 3 h. The liposome size was measured by diluting in 1× DPBS and performing DLS characterization using a Zetasizer (Malvern), and liposome concentration was determined using a phosphatidylcholine (PC) assay kit (Sigma). DPPC liposomes were kept at 45 °C until LSNA synthesis on the same day. siRNA was duplexed following a previously described hybridization method.²⁰ To form LSNAs, siRNA was added to DPPC liposomes at a mole ratio of 30 siRNA duplexes to 1 liposome at a final siRNA concentration of 500 nM. Higher siRNA duplex:liposome ratios did not result in any increase in siRNA duplex loading on the LSNA. The siRNA/liposome mixture was vortexed, sonicated for 30 s, then incubated with shaking at 45 °C overnight. Unattached oligonucleotides were removed by washing with 1× DPBS three times in Amicon Ultra 100K molecular weight cutoff spin filters (MilliporeSigma). The SNAs were stored at 4 °C for up to 1 month.

LSNA Characterization.

LSNA formation was confirmed using gel electrophoresis. 50 pmol equivalent of linear tocopherol-anchor hairpin-like siRNA or hairpin-like siRNA-LSNA was combined with 1 μL of purple gel loading dye (6×) without SDS (New England Biolabs) and brought up to a volume of 6 μL using 1× DPBS. The samples were run on a 1% agarose gel in 1× tris/borate/ethylenediaminetetraacetic acid (EDTA/TBE) on ice at 100 V for 1.5 h. The gels were imaged and analyzed using a ChemiDoc MP imaging system (Bio-Rad). LSNA size was measured by diluting in 1× DPBS and performing DLS using a Zetasizer.

OliGreen assay was used to quantify siRNA duplex loading on the hairpin-like siRNA-LSNA. 5 μL of LSNAs were diluted in 70 μL of 1× DPBS, then mixed with 75 μL of 0.2% Triton X-100 (Sigma) in 1× DPBS to dissociate the liposome core. 25 μL of this mixture was added to 75 μL of 1× DPBS and 100 μL of 0.5% Quanti-iT OliGreen reagent (Invitrogen) in 1× DPBS. The samples were then analyzed in a 96-well black, clear-bottom plate by measuring OliGreen fluorescence (λ_ex = 480 nm, λ_em = 520 nm) using a Cytation 5 imaging multi-mode reader (BioTek) and comparing to a standard curve of hairpin-like siRNA to determine siRNA concentration in the sample. The liposome concentration in the sample was determined using the previously mentioned PC assay and adjusted to desired working concentrations. siRNA duplex loading on the LSNA was then calculated by taking the ratio of hairpin-like siRNA concentration to liposome concentration.

PicoGreen assay was used to quantify siRNA duplex loading on the hybridized siRNA-LSNA. 5 μL of LSNAs were combined with 12 μL of RNase A/T1 mix (Thermo) to degrade lone passenger RNA strands, 58 μL of 1× DPBS, and 75 μL of 0.2% Triton X-100 in 1× DPBS to dissociate the liposome core. The mixture was incubated at 37 °C for 10 min to activate the RNase. Next, 25 μL of this mixture was added to 75 μL of 1× DPBS and 100 μL of 0.5% Quanti-iT PicoGreen reagent (Invitrogen) in 1× DPBS. The samples were then analyzed in a 96-well black, clear-bottom plate by measuring PicoGreen fluorescence (λ_ex = 480 nm, λ_em = 520 nm) using a Cytation 5 imaging multi-mode reader and comparing to a standard curve of hybridized siRNA to determine siRNA concentration in the sample. siRNA duplex loading of the hybridized siRNA-LSNA was then calculated by taking the ratio of hybridized siRNA concentration to liposome concentration.

AuSNA Synthesis and Characterization.

The synthesis and characterization of hairpin-like siRNA-AuSNAs were performed following a previously established protocol.²⁰

Flow Cytometry of HUVECs to Measure Uptake of LSNAs.

HUVECs (ATCC) were grown in Endothelial Cell Growth Medium-2 (EGM-2, Lonza) containing BulletKit growth factors. For cell uptake studies, HUVECs were seeded in a 24-well cell culture plate at 40,000 cells/well for 48 h. Cy5-labeled linear duplexed siRNA or hairpin-like siRNA-LSNAs were added to the cells at a concentration of 100 nM siRNA equivalent in EGM-2. At time points of 4 h and 24 h after administration, cells were washed with 500 μL of HEPES buffer, then detached by adding 150 μL Trypsin/EDTA and incubating at 37 °C for 5 min. Cells were transferred to flow cytometry tubes, neutralized with 200 μL EGM-2, and subsequently pelleted by centrifugation. To stain for live cells, cells were resuspended in 100 μL HEPES buffer containing 0.5 μL live/dead fixable blue reactive dye (Invitrogen), then incubated for 15 min at room temperature. Next, 600 μL of HEPES buffer was added, then the cells were spun down and the supernatant was removed. The cells were resuspended in 150 μL of fixation buffer (BioLegend) and stored at 4 °C until analyzed. Flow cytometry was performed using an A3 Symphony flow cytometer (BD Biosciences) with data analysis performed using FlowJo v. 10.

Confocal Microscopy of HUVECs to Observe Uptake of LSNAs.

HUVECs were seeded in an 8-well slide plate at 40,000 cells/well for 24 h. Cy5-labeled linear duplexed siRNA or hairpin-like siRNA-LSNAs were added to the cells at a concentration of 100 nM siRNA equivalent in EGM-2. At 4 h after administration, the cells were washed with 200 μL of 1× DPBS and subsequently fixed with 150 μL fixation buffer (BioLegend) for 15 min at 4 °C. Cells were then washed with 200 μL PBS and subsequently stained for 1 min using 300 nM 4′,6-diamidino-2-phenylindole (DAPI) in 1× DPBS to label cell nuclei. After staining, the cells were briefly washed with 200 μL of 1× DPBS and stored in 1× DPBS until imaged. Confocal microscopy was performed using a Zeiss LSM 800 confocal laser scanning microscope.

Cytotoxicity of SNAs.

Hairpin-like siRNA-AuSNAs were synthesized and characterized following a previously described method.²⁰ HUVECs were seeded in a 96-well cell culture plate at 2,500 cells/well for 24 h. Hairpin-like siRNA-AuSNAs and hairpin-like siRNA-LSNAs in EGM-2 at a range of concentrations were added to the cells for 48 h. The wells were washed with 1× DPBS 3 times to remove dead cells and SNAs that were not internalized by cells. Next, 50 μL of 1× DPBS and 50 μL of CellTiter-Glo 2.0 reagent (Promega) was added to the wells and incubated in the dark for 10 min. Luminescence was measured using a BioTek Cytation 5 imaging reader as an indicator of live cell count. Cell viability was normalized to cells treated with EGM-2 only.

Tube Formation Assay.

Phenol-red free Matrigel basement membrane matrix (Corning) was thawed overnight in the fridge. Next, 75 μL of thawed Matrigel was added to the wells of a pre-cooled 96-well clear cell culture plate on ice using pre-cooled 200 μL pipette tips. The plate was then taken off ice and incubated at room temperature for 10 min, then at 37 °C for 30 min to solidify the Matrigel. For treatments, 30,000 HUVECs were combined with nontargeting hairpin-like siRNA-LSNAs or VEGF-targeting hairpin-like siRNA-LSNAs in EGM-2 in a total volume of 75 μL and incubated at 37 °C for 1 h. The treated cells were then added to the Matrigel and kept at 37 °C. Images of cells in Matrigel were captured using an Eclipse Ts2 inverted microscope (Nikon) at 4× magnification at 24 h and 48 h after treatment. Tube formation was analyzed and quantified using ImageJ and the Angiogenesis Analyzer tool.⁴⁰

Statistics.

All statistical analyses were performed using GraphPad Prism. For comparisons between two groups, means were compared using an unpaired, two-tailed t-test. For comparisons between more than two groups, means were compared using an ordinary one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. P values were multiplicity adjusted to account for multiple comparisons. Cytotoxicity results were fit with a 3-parameter logistic curve using a least-squares fit.