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. 2024 Aug 15;7(9):2840–2855. doi: 10.1021/acsptsci.4c00336

Lipopolymer/siRNA Nanoparticles Targeting the Signal Transducer and Activator of Transcription 5A Disrupts Proliferation of Acute Lymphoblastic Leukemia

Mohammad Nasrullah †,, Remant KC , Kyle Nickel , Kylie Parent , Cezary Kucharski , Daniel Nisakar Meenakshi Sundaram , Amarnath Praphakar Rajendran , Xiaoyan Jiang §, Joseph Brandwein , Hasan Uludağ †,‡,*
PMCID: PMC11406681  PMID: 39296267

Abstract

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The therapeutic potential of small interfering RNAs (siRNAs) in gene-targeted treatments is substantial, but their suboptimal delivery impedes widespread clinical applications. Critical among these is the inability of siRNAs to traverse the cell membranes due to their anionic nature and high molecular weight. This limitation is particularly pronounced in lymphocytes, which pose additional barriers due to their smaller size and scant cytoplasm. Addressing this, we introduce an innovative lipid-conjugated polyethylenimine lipopolymer platform, engineered for delivery of therapeutic siRNAs into lymphocytes. This system utilizes the cationic nature of the polyethylenimine for forming stable complexes with anionic siRNAs, while the lipid component facilitates cellular entry of siRNA. The resulting lipopolymer/siRNA complexes are termed lipopolymer nanoparticles (LPNPs). We comprehensively profiled the efficacy of this platform in human peripheral blood mononuclear cells (PBMCs) as well as in vitro and in vivo models of acute lymphoblastic leukemia (ALL), emphasizing the inhibition of the oncogenic signal transducer and activator of transcription 5A (STAT5A) gene. The lipopolymers demonstrated high efficiency in delivering siRNA to ALL cell lines (RS4;11 and SUP-B15) and primary patient cells, effectively silencing the STAT5A gene. The resultant gene silencing induced apoptosis and significantly reduced colony formation in vitro. Furthermore, in vivo studies showed a significant decrease in tumor volumes without causing substantial toxicity. The lipopolymers did not induce the secretion of proinflammatory cytokines (IL-6, TNF-α, and INF-γ) in PBMCs from healthy volunteers, underscoring their immune safety profile. Our observations indicate that LPNP-based siRNA delivery systems offer a promising therapeutic approach for ALL in terms of both safety and therapeutic efficacy.

Keywords: lipopolymer, siRNA delivery, STAT5A, acute lymphoblastic leukemia, nanoparticle, nonviral gene therapy


Small interfering RNA (siRNA), a key tool in biomedical research for loss-of-function studies, also emerged as a potential therapeutic avenue in cancer treatment by harnessing RNA interference (RNAi) mechanisms to precisely target and silence oncogenes, thus curbing the growth of cancer cells. However, translating it from “theoretical” potential to clinical application has been obstructed by significant biological barriers. Naked siRNA, without any protective measures, is highly susceptible to enzymatic (nuclease) degradation in the bloodstream, considerably declining its therapeutic efficiency.1 The features of siRNA, both its large molecular weight (>14 kDa) and highly anionic nature, further complicate cellular uptake, preventing effective delivery into target cells.2 Upon cellular entry, siRNA molecules often find themselves trapped within endosomes, preventing them from reaching their intended intracellular targets. This entrapment, combined with rapid systemic clearance, potential immunogenic responses, challenges in avoiding off-target effects, and difficulties in achieving specific tissue localization, emphasizes the need for advanced siRNA delivery systems.3 These systems must encapsulate and protect siRNA from degradation, enhance cellular uptake, facilitate endosomal escape, and minimize immune responses and off-target interactions. Delivering siRNA to primary cells such as peripheral blood mononuclear cells (PBMCs) presents an additional set of challenges. These include inherent resistance to transfection due to complex intracellular barriers, immune sensitivity to vectors/siRNAs, slower and more variable division rates, and a complex gene regulation environment.4,5 Moreover, lymphoblast-type cells are considered hard-to-transfect due to their smaller size, scant cytoplasm and limited endocytotic activity.6 Thus, advancing siRNA-based therapeutic strategies for health conditions stemming from lymphoblast-type cells, such as acute lymphoblastic leukemia (ALL), presents a significant challenge, despite the clinical promise of siRNA therapies, evidenced by the US FDA’s approval of six siRNA-based drugs.7

ALL is a common hematological malignancy, prevalent in children and characterized by the aberrant proliferation and accumulation of malignant lymphoid progenitor cells in the bone marrow milieu. Despite advancements in treatment, ALL continues to be a leading cause of cancer-related deaths in children, with poor outcomes in adults, as only 30% achieve long-term disease-free survival,8,9 largely due to the nonspecific nature of current therapies.10 The microenvironment of the bone marrow has been shown to provide a protective niche for leukemia cells, which further complicates ALL inhibition approaches. Genetic mutations and chromosomal rearrangements play crucial roles in ALL development.11 A crucial factor in ALL pathology is the constitutive activation of the signal transducer and activator of transcription 5A, or STAT5A, a transcription factor involved in immune regulation and cell proliferation.12 Caused by upstream tyrosine kinase mutations, this activation promotes leukemia by upregulating downstream genes like A1, PIM1, and BCL2(1316) and leads to increased cell proliferation, survival, drug resistance, and the suppression of apoptosis in ALL.14,1719 Inhibiting STAT5A has shown potential in reducing leukemogenesis in ALL.20 Challenges associated with current therapies, such as relapses and refractory cases,21 highlight the urgent need for more effective and safer treatments for ALL, aiming to achieve more durable, ideally complete, remissions.

Addressing these challenges, our research explored a nonviral siRNA delivery system based on lipopolymers to enable siRNA therapy of leukemia. By conjugating aliphatic lipids onto branched polyethylenimine (PEI), we developed three different lipopolymers, named here as PEI-A, PEI-B, and PEI-C. These lipopolymers form complexes with siRNA, known as lipopolymer nanoparticles (LPNPs), which protect the siRNA from degradation and enhance its delivery and intracellular release. Harnessing the electrostatic interactions provided by PEI’s repeating amine groups and enhanced through lipid grafting, these lipopolymers have shown promise in delivering siRNA to various cancer cell lines. Our prior investigations have extensively characterized these lipopolymers across various cancer models, including attachment cell lines from breast cancer,2224 non-ALL leukemia cell lines like acute myeloid leukemia cells2527 and chronic myeloid leukemia cells,2831 and in vitro models of ALL.16 Building upon this foundational research, this study seeks to assess the safety and therapeutic efficacy of these lipopolymers within a preclinical model of ALL by targeting STAT5A. Through assessing the safety and effectiveness of our LPNPs both in vitro and in vivo, including experiments on primary cells from human healthy volunteers, ALL cell lines, and primary cells from ALL patients, we confirmed these lipopolymers as effective siRNA carriers, offering a new avenue for developing targeted therapies for ALL.

Materials and Methods

Materials

The following siRNAs were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA): STAT5A siRNA (siSTAT5A) and scrambled control siRNA (CsiRNA) (IDT) (Table S1). Fluorescein (FAM)-labeled scrambled siRNA (FAM-siRNA) was also from IDT and used to visualize and quantify the cellular uptake of siRNA. Three lipopolymers used in this study were derived from PEI, named as PEI-A, PEI-B, and PEI-C, and were obtained from RJH Biosciences (Edmonton, Alberta). Primers for human β-actin, STAT5A, and the cytokines (interleukin 6, IL-6; interferon-γ, IFN-γ; tumor necrosis factor-α, TNF-α) used in RT-qPCR analysis were obtained from IDT (Table S2). Validated DUO ELISA kits (cat. no. DY210-05: human TNF-α, cat. no. DY206-05: human IL-6, cat. no. DY285B-05: human IFN-γ, and cat. no. DY008B: DuoSet ELISA ancillary reagent kit 2) were obtained from R&D Systems. The apoptosis kit (FITC-annexin V/PI assay) was purchased from BD Biosciences (cat. no. 556547). Methylcellulose was obtained from R&D Systems, Inc. (cat. no. HSC005). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was obtained from Alfa Aesar (Ward Hill, MA).

Cells and Culture

Eleven fresh peripheral blood samples from healthy volunteers were obtained from the Canadian Blood Services (ethics approval secured by the University of Alberta and Canadian Blood Services Ethics Boards). Peripheral blood mononuclear cells (PBMCs) were isolated with the help of Ficoll-Paque PLUS media. In brief, 15 mL of Ficoll-Paque PLUS was added to a 50 mL tube. Then, buffy coats were diluted with an equal volume of HBSS, and 20 mL of this diluted sample was carefully layered over Ficoll-Paque PLUS. The mixture was then centrifuged at 400g for 40 min at room temperature. Without disturbing the interface lymphocyte layer, the upper plasma layer was discarded, and the lymphocytes were collected in a fresh tube. HBSS (3× volume) was added to the lymphocytes and centrifuged again at 100g for 10 min at room temperature (3 times). The supernatant was discarded carefully. Pellets (PBMCs) were suspended in the MN culture medium for culturing. Fresh PBMCs were used for further studies.

Two ALL cell lines (RS4;11 and SUP-B15) were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). RS4;11 cells were cultured in an RPMI 1640 medium supplemented with 10% FBS and 100 U mL–1 penicillin and 100 μg mL–1 streptomycin. SUP-B15 cells were cultured in IMDM supplemented with 20% FBS, 0.05 mM 2-mercaptoethanol, 100 U mL–1 penicillin, and 100 μg mL–1 streptomycin. Both cell lines were cultured at 37 °C and 5% CO2 for maintaining. Cells were subcultured after reaching 80% confluency. For subculture, cells were centrifuged for 5 min at 600 rpm and passaged at a 20% concentration of the original density.

Frozen samples of three ALL patients were obtained from the Biopathology Center of the Children’s Oncology Group (Philadelphia, PA). Ethical approval was obtained from the University of Alberta’s Health Research Ethics Board before the receipt of samples. Frozen cryovials were thawed partially (not complete dissolving) to culture the mononuclear (MN) cells. Then, thawed cells were carefully added to 4 mL of the recovery medium dropwise and incubated for 2–4 min at room temperature to get rid of any clumps. Suspended MN cells were then spin down at 500 rcf for 10 min at 4 °C. Supernatants were carefully removed, and pellets were resuspended in the MN culture medium, which consisted of an IMDM serum-free medium (80% v/v), a BIT 9500 serum substitute (20% v/v), GlutaMAX (2 mM), IL-3 (10 ng/mL), IL-6 (10 ng/mL), IL-7 (10 ng/mL), Flt3-ligand (20 ng/mL), SCF (30 ng/mL), and 2-mercaptoethanol (0.1 mM)

LPNP Preparation, Characterization, and Transfection

Lyophilized siRNAs were diluted with RNase-free water to prepare the stock solution according to the manufacturer’s guideline. Working concentrations (0.14 μg/μL) of siRNA were prepared by a dilution buffer (IDT). Lipopolymers (PEI-A, PEI-B, and PEI-C) were diluted to a working concentration of 1 μg/μL. Lipopolymer/siRNA complexes were prepared by incubating siRNA, lipopolymers, and RPMI 1640 (serum-free) together in a tube for 30 min. The amount of the siRNA used in formulations was calculated based on the final desired concentration (e.g., 60 nM) of siRNA for cell treatment, and the amount of the lipopolymers was calculated based on the weight ratio of lipopolymer/siRNA (e.g., 5.0 and 7.5, Table S3 and Figure S1).

The siRNA binding efficiency of lipopolymers was assessed using the SYBR Green I dye (Cambrex BioScience, Rockland, MD) exclusion method. Solutions of lipopolymers were adjusted with ddH2O to concentrations ranging from 0 to 0.056 μg/μL. Then, 2 μL of CsiRNA (0.14 μg/μL in water) was added to these solutions followed by vortexing to create complexes across various weight/weight (w/w) ratios. After 30 min at room temperature, complexes were mixed with SYBR Green I (1× in a TAE buffer), transferred to a black 96-well plate, and analyzed for fluorescence to measure free siRNA levels, and fluorescence was read in a fluorometer at an excitation/emission wavelength of 485/527 nm (Fluoroskan Ascent, Thermo Labsystems). The binding capability of lipopolymers was quantified as the BC50 value, indicating that the lipopolymer-to-siRNA ratio needed to bind half of the siRNA.

For physical characterization, lipopolymers were complexed with CsiRNA in an aqueous medium and tested with a Litesizer 500 (Anton Paar, GmbH). Briefly, after preparing complexes and incubating for 30 min at room temperature, complexes were diluted to a final volume of 1000 uL with RNase-free water. The diluted samples were transferred into Omega cuvettes to measure the size and ζ potential. The instrument was set at a 175° backscatter and a temperature of 25 °C with the solvent as water. Transmission electron microscopy (TEM) was also used to examine the complexes, following the previously mentioned preparation method.32 A 10 μL drop of the solution was placed on a carbon-coated copper TEM grid and left to air-dry for 10 min at room temperature. The grid was then stained using 5 μL of 1% (w/v) uranyl acetate and allowed to dry naturally overnight. The structural appearance of the complexes was observed using an FEI TEM with a Gatan camera.

For all biological experiments, cells were treated with a reverse transfection protocol. LPNPs were transferred to the cell culture plates first, and the desired number of cells was seeded to the culture plates and incubated at 37 °C. After incubating at the experimentally designed time point, cells were harvested for further analysis.

Cytokine Response to LPNPs

The PBMCs from healthy volunteers were transfected with LPNPs to determine cytokine secretion upon siRNA treatment; 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 5 μg/mL ionomycin (IO) were used alone or in combination (PMA+IO) as positive controls for chemical stimulators of cytokine secretion. After 24 h of incubation, the supernatants were collected and frozen for analysis by validated ELISA kits for the proinflammatory cytokines, human TNF-α, IL-6, and IFN-γ, by following the manufacturer’s protocol. The cells were then collected to assess the mRNA expression levels of proinflammatory cytokines using real-time quantitative PCR (RT-qPCR). The total RNA was isolated by a TRIzol reagent (Invitrogen, Carlsbad, CA) and stored until further analysis.

Analysis of Gene Expression

The success of targeted gene silencing, induced through lipopolymer-mediated siRNA delivery, was measured by using RT-qPCR analysis. The quantity and purity of isolated RNA were determined using a Nanodrop Lite spectrophotometer (Thermo Fisher). The cDNA was synthesized from total RNA using a SensiFAST cDNA synthesis kit (cat no. BIO-65054, Meridian Life Science, Inc.). RT-qPCR analysis was then performed with 100 ng of cDNA in a StepOnePlus real-time PCR system (Applied Biosystems), using a SensiFAST SYBR Hi-ROX master mix (cat no. BIO-92005, Meridian Life Science, Inc.). ROX was used as the passive reference dye, and the sequence of specific primers used for the intended analysis is summarized in Cells and Culture. The PCR cycle used was as follows: 95 °C for 10 min, 40 cycles of 95 °C for 10 s, and 65 °C for 30 s. Following the PCR cycle, melt-curve analysis was performed as follows: 95 °C for 15 s, then 60 °C for 1 min, followed by a ramp rate of +0.3 °C s–1 to 95 °C. The ΔΔCt values were determined using β-actin as a housekeeping gene. Gene expression levels were reported as the quantity of transcripts relative to the untreated group and compared with the CsiRNA group.

Cellular Uptake of siRNA

To determine the efficacy of siRNA delivery into the cells, lipopolymers were complexed with FAM-siRNA at LPNP ratios of 5/7.5/10 to prepare complexes (method described previously). RS4;11 and SUP-B15 cells were transfected with these complexes in a 48-well plate (final siRNA concentration of 60 nM in a medium) and incubated at 37 °C. Untreated cells or cells treated with naked siRNA (no LPNPs) were used as controls. After 24 h of incubation, cells were transferred to microcentrifuge tubes for centrifugation (1800 rpm for 8 min). The cells were washed twice with HBSS (pH 7.4) and resuspended in formaldehyde (3.7% in HBSS) for 30 min to fix the cells. The cellular uptake of FAM-siRNA by complexes was quantified by a BD LSR Fortessa-SORP flow cytometer (BD, Biosciences, Frankin Lakes, NJ). The FAM-siRNA delivery was summarized as the mean fluorescence intensity of the recovered cell population and the percentage of cells showing FAM-siRNA fluorescence.

The cellular uptake of the same complexes was also visualized by microscopy in both cell lines. In brief, FAM-siRNA was transfected into cells with the help of lipopolymers and incubated at 37 °C. Twenty-four hours postincubation, the cells were washed with HBSS solution, fixed with formaldehyde (3.7% in HBSS) for 30 min, and stained with DAPI (Brunschwig Chemie, Amsterdam, The Netherlands) to visualize the nuclear border. Then, the images of FAM-labeled cells were acquired under a fluorescence microscope (OLYMPUS FSX100, Olympus America, Center Valley, PA). FAM-labeled cells were compared with untreated or naked siRNA transfected cells to evaluate the siRNA delivery efficiencies of the lipopolymers.

Apoptosis Assay

The percentages of apoptotic cells in the treatment groups were assessed by using the FITC-annexin V and propidium iodide (PI) staining protocol. In brief, after the RS4;11 and SUP-B15 cells were transfected with the LPNPs for 3 days, cells were harvested, washed with HBSS, and transferred into the apoptosis binding buffer supplied by the manufacturer. They were then treated with FITC-annexin V and propidium iodide (PI) and incubated in the dark for 15 min at room temperature. Finally, the number of apoptotic cells from treatment groups (FITC-annexin V+, PI+) was analyzed using a BD LSR Fortessa-SORP flow cytometer (BD, Biosciences, Frankin Lakes, NJ) and compared with untreated cells.

Colony Forming Unit (CFU) Assay

The CFU assay was used to evaluate the cytotoxicity and cellular growth inhibitory effects of investigational targeted therapies in the suspension cells. RS4;11 and SUP-B15 cells were transfected with LPNPs as described before and incubated for 24 h. Cells were then counted using Trypan Blue cell viability staining and a hemocytometer to determine the number of viable cells. A total of 1500 RS4;11 cells or 5000 SUP-B15 cells were then mixed with 500 μL of human methylcellulose-enriched media (1.4%) and grown in 24-well plates for 14 days at 37 °C with 5% CO2. After 14 days of incubation, the total number of colonies was observed and counted manually under a microscope. Inhibition of colony formation in siSTAT5A-treated cells was compared with CsiRNA-treated cells relative to the untreated cells. After counting, colonies were stained with 100 μL of MTT solution (5 mg/mL) and incubated at 37 °C for 3 h before taking corresponding images.

Animal Studies

Animal experiments were performed following procedures preapproved by the Health Sciences Laboratory Animal Services (HSLAS), University of Alberta. Triple-immunodeficient male NOD-Prkdcem26Cd52Il2rgem26Cd22 (NCG) mice (male, 6–8 weeks) were obtained from Charles River Laboratories (Laval, QC, Canada). RS4;11 cells (2.5 × 106 cells suspended in 100 μL of 60:40% RPMI:Matrigel) were administered subcutaneously (SQ), and sufficient time was given for the establishment of ALL xenografts in mice. The tumor volume was measured every 3 days, and animals were recruited to the study when the tumor size reached >100 mm3. The tumor volume was determined by using measurements from a digital caliper. The formula used to calculate the tumor volume (TV) involved measuring the length (L) and width (W) of the tumor, with the calculation defined as TV = (L × W2)/2. siRNAs (25 μg) complexed with PEI-C (1 mg/kg body weight of mouse) were injected SQ every 3 days for 5 times. The mice were typically monitored within 24 h postinjection. After 3 days of the final dose, mice were euthanized, and tumors were collected for isolating RNA with TRIzol and further analysis of STAT5A expression by RT-qPCR (Figure 7A).

Figure 7.

Figure 7

Effects of siSTAT5A LPNPs on the ALL xenograft model. (A) Flowchart depicting the in vivo study involving RS4;11 xenografts in NCG mice. The siSTAT5A or CsiRNA was complexed with PEI-C and injected subcutaneously 5 times at a 1 mg/kg (siRNA) dose. (B) Differences in the relative tumor volume between the CsiRNA- and siSTAT5A-treated mice increased with time (mean + SD, n = 6 or 7 mice at each time point). Significances were analyzed by two-way ANOVA and Sidak’s multiple comparisons test (*p = 0.0261). (C) Changes in the body weight of mice over the study duration. Bars show means + SD. (D) Changes in STAT5A mRNA in tumor samples were analyzed by normalizing the gene to a reference gene (β-actin). Significance was determined by using a parametric unpaired t-test. Data are presented as the mean + SD (**p = 0.0073). SQ, subcutaneous.

For in vivo biocompatibility of PEI-C/siRNA complexes, 6–8 weeks healthy male BALB/C mice (Charles River Laboratories, Laval, QC, Canada) were SQ injected with either a sham solution (RPMI) or PEI/CsiRNA complexes (1 mg/kg of siRNA). For blood cell counts, the mice received a single SQ injection. After 24 h, whole blood was collected via cardiac puncture following humane euthanization. For the serum biochemistry profile, the mice were given SQ injections every third day for a total of 5 times. After 24 h of the final injection, whole blood was collected via cardiac puncture, and the serum was obtained by immediate centrifugation at 4 °C for further analysis. The Idexx Reference Laboratories Ltd. (Edmonton, AB, Canada) analyzed both whole blood and serum samples.

Statistical Analysis

All statistical analysis methods are described in the figure legends. Statistical analysis was performed with GraphPad Prism 8.0.2 or Excel Worksheet (Microsoft 365). Data were presented as the mean + SD with at least three technical replicates unless otherwise mentioned. The statistical tests used to calculate p-values are listed in the figure legends, with significances considered when p < 0.05.

Results

Characterization of LPNPs

In this study, we utilized three lipopolymer carriers derived from conjugating different lipids to 1.2 kDa PEI, named PEI-A, PEI-B, and PEI-C, and evaluated their effectiveness in ALL cell transfection, after a comprehensive screening of lipopolymer libraries (not shown). Initially, we assessed the capacity of the selected carriers to bind siRNA, which is essential for the effective delivery of siRNA into the cells. All three lipopolymers demonstrated similar binding capabilities as a function of lipopolymer/siRNA (w/w) ratios (Figure 1A,B). We also determined the hydrodynamic sizes and ζ potentials of the LPNPs across three different lipopolymer/siRNA (w/w) ratios (5.0, 7.5, and 10.0). The results indicated that PEI-B and PEI-C complexes exhibited relatively fewer variations in hydrodynamic sizes when shifting from lower to higher ratios, although a general trend of decreasing sizes with increasing lipopolymer/siRNA ratios was observed. In contrast, an increase in the ratio for PEI-A led to a doubling of the hydrodynamic size with average sizes exceeding 400 nm for the lipopolymer/siRNA ratio of 10. At the intermediate ratio of 7.5, the hydrodynamic sizes were measured as 291.5 nm for PEI-A, 121.9 nm for PEI-B, and 168.4 nm for PEI-C (Figure 1C and Figure S2), and this ratio was chosen for subsequent experiments. Elevating the lipopolymer/siRNA ratio from 5.0 to 10.0 resulted in an increase in the zeta potentials of the complexes, albeit at a variable for each carrier: from 9.1 to 16.2 mV for PEI-A, from 12.8 to 15.7 mV for PEI-B, and from 19.1 to 19.6 mV for PEI-C (Figure 1D). Notably, PEI-C exhibited a higher ζ potential at a ratio of 7.5 compared to a 10 ratio, with a zeta potential of 21.63 mV. The TEM images of nanocomplexes with a w/w ratio of 7.5 are shown in Figure 1E. TEM micrographs of the complexes closely matched the hydrodynamic sizes measured by the Litesizer, with both techniques indicating that the PEI-A complex was large among the study groups.

Figure 1.

Figure 1

Characterization of complexes between lipopolymers PEI-A, PEI-B, and PEI-C and siRNA. (A) siRNA binding capacity of the lipopolymers. (B) Lipopolymer/siRNA formulation ratio (w/w) required for binding 50% of the siRNA (BC50). (C) Hydrodynamic size of LPNPs formulated at different ratios (5.0:1.0, 7.5:1.0, and 10.0:1.0). (D) Surface charge (measured as the ζ potential) of the same complexes. (E) TEM images of LPNPs prepared at a ratio of 7.5:1.0.

LPNP Delivery Avoids Cytokine Response and Reduces STAT5A Expression in Human PBMCs

Prior to exploring the therapeutic potential of LPNPs in treating ALL, it was essential to first determine if the lipopolymers could stimulate production of proinflammatory cytokines, particularly IL-6, IFN-γ, and TNF-α, given that lipid-based siRNA therapies are known to potentially induce unwanted immune reactions.33 We isolated PBMCs from human healthy volunteers using Ficoll-Paque PLUS and transfected the cells with siSTAT5A/CsiRNA LPNPs. Cells were stimulated with chemical stimulants PMA and IO as a reference, while the complexes were setup at the lipopolymer/siRNA ratios of 7.5 (previously used) and 12, which represents an excess of lipopolymers. The siRNAs used were CsiRNA and siSTAT5A on their own or as complexes of CsiRNA with the three chosen carriers. After 24 h of transfection, cytokine response was measured by RT-qPCR (gene expression) and ELISA (protein secretion). The PMA/IO combination provided the highest stimulation of cytokine gene expression (Figure 2B) and cytokine protein secretion (Figure 2C). In contrast, siRNAs alone did not significantly stimulate cytokine production compared with the PMA/IO combination. Overall, the LPNPs elicited minimal or no cytokine response, regardless of the formulation (Figure 2D–I). Specifically, PEI-A and PEI-B showed minimal effects on IL-6 gene expression and protein secretion. However, PEI-C/siSTAT5A at R12.0 induced relatively higher IL-6 gene expression (Figure 2H), but this was not accompanied by increased protein secretions in the supernatant, likely due to post-transcriptional regulation, rapid intracellular degradation, or secretion pathway inefficiencies. For TNF-α, neither the lipopolymer alone nor the complexes with siRNA induced gene expression or protein secretion in PBMCs, indicating that both the lipopolymer and its siRNA complexes do not activate this cytokine. Compared to the other cytokines, a modest increase in IFN-γ gene expression and protein secretion was observed with the lipopolymer alone or its siRNA complexes. This modest increase can be explained by the relatively high baseline secretion of IFN-γ in the untreated group (13.1 pg/mL), as well as in the siRNA alone-treated groups, CsiRNA (14 pg/mL) and siSTAT5A (26 pg/mL) (Figure 2C). Higher IFN-γ secretion was observed with LPNPs, specifically in the PEI-B/siSTAT5A at the R7.5 group (44.33 pg/mL, Figure 2G) and the PEI-C/siSTAT5A at the R7.5 group (23.1 pg/mL, Figure 2I). Notably, IFN-γ levels were also higher in the untreated group (13.11 pg/mL) compared to IL-6 (0.86 pg/mL) and TNF-α (4.67 pg/mL). This moderate IFN-γ secretion is unlikely to have adverse biological consequences, as indicated by findings from Castelhano and colleagues, who reported IFN-γ levels up to 125.54 pg/mL in supernatants from 24 h cultured PBMCs of 20 healthy human volunteers without stimulation.34 Moreover, these increases in IFN-γ secretion were minimal compared to the high levels observed with the PAM/IO combination treatments.

Figure 2.

Figure 2

Proinflammatory cytokine response to LPNPs in human PBMCs. (A) Schematic diagram (created with BioRender.com) of PBMC isolation from peripheral blood of human healthy volunteers and treatment with positive controls or LPNPs at a regular ratio of 7.5:1.0 (R7.5, w/w) and a higher ratio of 12.0:1.0 (R12.0, w/w). After 24 h of treatment, cell pellets were collected for RT-qPCR, and culture supernatants were collected for ELISA. (B,D,F,H) Proinflammatory cytokine (IL-6, IFN-γ, and TNF-α) mRNA levels in PBMCs by positive controls (PMA/IO/PMA+IO) and siRNAs alone (60 nM CsiRNA/siSTAT5A) (B), PEI-A alone or complexed with siRNAs (D), PEI-B alone or complexed with siRNA (F), and PEI-C alone or complexed with siRNAs (H). (C,E,G,I) Protein (IL-6, IFN-γ, and TNF-α) levels in culture supernatants in PBMCs by positive controls (PMA/IO/PMA+IO), siRNAs alone (60 nM CsiRNA/siSTAT5A) (C), PEI-A alone or complexed with siRNAs (E), PEI-B alone or complexed with siRNAs (G), and PEI-C alone or complexed with siRNAs (I). UT: untreated cells.

We next explored the capacity of our complexes for gene silencing, specifically targeting STAT5A, in primary cells obtained from a cohort of 11 healthy human volunteers (HVs). The STAT5A is constitutively activated in ALL, and it is known as a hallmark for this malignancy.3537 It is also regularly activated by various cytokines or growth factors in healthy humans and plays essential roles in a variety of biological processes, including the generation and regulation of immune cells as well as hematopoiesis.38,39 We sought to assess the ability of our lipopolymers to effectively transfect PBMCs to downregulate STAT5A expression. The transfection of PBMCs with lipopolymer/siSTAT5A complexes yielded heterogeneous results, with variations in the extent of STAT5A silencing among the samples. No significant STAT5A silencing was seen in the two samples, HV08 and HV09, with any of the lipopolymers. Among the remaining 9 HV samples, differential responses to three distinct lipopolymer formulations were recorded: 2 samples showed significant responses to PEI-A, 6 samples to PEI-B, and 8 samples to PEI-C (Table S4). Upon analyzing the average STAT5A silencing data from samples of 11 HVs (Figure 3, bottom right graph), it was observed that the complexes formed by PEI-B/siSTAT5A achieved a substantial 56.8% knockdown of STAT5A, which was the maximum among the three evaluated lipopolymers. In comparison, PEI-C/siSTAT5A complexes resulted in a 40.2% knockdown, whereas PEI-A/siSTAT5A complexes achieved only a 12.7% knockdown.

Figure 3.

Figure 3

STAT5A gene silencing by LPNPs in human PBMCs. PBMCs were harvested from 11 healthy volunteers and transfected with LPNPs in a ratio of 7.5:1.0. After 3 days of incubation, RT-qPCR analysis was conducted for STAT5A expression. The figure displays downregulation of STAT5A expression individually in all samples and the average silencing across all samples (bottom right graph). Data are shown as the mean + SD. Significances were compared with CsiRNA and analyzed by two-way ANOVA (Sidak’s multiple comparisons test). ***p < 0.0002, **p < 0.002, and *p < 0.03. HV, healthy volunteer.

Efficient siRNA Delivery to ALL Cells by Lipopolymers

To explore the specific utility of the complexes in ALL treatment, cellular uptake of FAM-labeled siRNAs complexed with the lipopolymers was investigated in 2 ALL cell lines, RS4;11 and SUP-B15, by using flow cytometry. These siRNAs were complexed at three distinct lipopolymer/siRNA ratios (2.5, 5.0, and 7.5). The analysis revealed a marked increase in siRNA uptake in both cell lines when complexed with the lipopolymers, as opposed to cells transfected with the naked siRNA. Notably, PEI-C was identified as the most efficient lipopolymer, showing the highest proportion of FAM-positive cells in RS4;11 cells (Figure 4 A,B), which is corroborated in SUP-B15 cells as well (Figure 4 C,D). Further analysis of the cellular uptake through fluorescence imaging revealed a pronounced difference in siRNA delivery; cells transfected with naked siRNA exhibited no detectable FAM-labeled siRNAs, whereas those transfected with LPNPs displayed clearly visible FAM-positive cells (Figure 5E). Consistent with the flow cytometry findings, PEI-C was the most effective at delivering siRNA in fluorescence microscopy as well, as shown by the clear visibility of FAM-positive cells in the imaged cells.

Figure 4.

Figure 4

Cellular uptake of FAM-siRNA complexes with lipopolymers in ALL cells. LPNPs were formulated at different ratios (2.5:1.0, 5.0:1.0, and 7.5:1.0 w/w) and transfected in ALL cell lines, RS4:11 (upper panel) and SUP-B15 (lower panel). (A,C) Flow cytometry results of the FAM-positive population. (B,D) Flow cytometry results of mean fluorescence intensity of the positive population. (E) Fluorescence microscope images of lipopolymers/FAM-siRNA complexes at a ratio of 7.5:1.0 (w/w) in both cell lines. UT, untreated cells.

Figure 5.

Figure 5

Silencing efficiency and antiapoptotic effects of siSTAT5A LPNPs on ALL cells in vitro. RS4;11 (left panel) and SUP-B15 (right panel) cells were treated with LPNPs in a ratio of 7.5:1.0. (A,B) STAT5A mRNA transcript levels were quantified by RT-qPCR 3 days after treatment (n = 3). (C,D) Flow cytometry analysis of apoptosis (FITC-annexin V and PI) 3 days after treatment (n = 3). Data are shown as the mean + SD. Significances were compared with CsiRNA and analyzed by two-way ANOVA (Sidak’s multiple comparisons test). ***p < 0.0002, **p < 0.002, and *p < 0.03. UT, untreated cells.

siSTAT5A LPNP Effectively Silences STAT5A, Induces Apoptosis, and Inhibits Colony Formation in ALL Cells

Following the successful cellular uptake of siRNA in ALL cells, we next explored whether the siRNAs delivered via lipopolymers can effectively downregulate target oncogene STAT5A. To this end, both RS4;11 and SUP-B15 cells were transfected with LPNPs, and the cells were harvested to analyze STAT5A knockdown through RT-qPCR 3 days post-transfection. The results from the RT-qPCR analysis demonstrated that all three lipopolymer formulations were successful in achieving the downregulation of STAT5A expression in both cells (Figure 5A,B). The induction of apoptosis in these cells by siSTAT5A LPNPs was then investigated via FITC-annexin V/PI staining and flow cytometry, revealing an increase in apoptotic cell populations in both cell types compared to untreated cells (Figure 5C,D and Figure S3). The percentages of apoptotic cells were calculated by subtracting the apoptotic cell values of the untreated group from those of the LPNP-treated groups for both cell lines. This evidence supports the efficacy of lipopolymer-delivered siRNAs in not only targeting and downregulating crucial oncogenes such as STAT5A but also in inducing apoptotic pathways within ALL cells.

We further evaluated the consequences of STAT5A silencing on inhibiting CFUs in the RS4;11 and SUP-B15 cells. We observed significant inhibition of colony formation in both cell types when treated with all three lipopolymer formulations (Figure 6). PEI-C achieved greater inhibition in RS4;11 cells (Figure 6B), while PEI-A was more inhibitory with SUP-B15 cells (Figure 6D). However, the PEI-A showed cytotoxicity when complexed with the CsiRNA (given by the lower percentage of CFU numbers in SUP-B15 cells, which is normalized against nontreated cells), whereas PEI-C showed no or minimal toxicity with its CsiRNA complexes. This reduction in the colony formation capacity highlights the broader potential of curbing ALL progression by LPNPs.

Figure 6.

Figure 6

Inhibitory effects of siSTAT5A LPNPs on ALL cells in vitro. RS4;11 (upper panel) and SUP-B15 (lower panel) cells were treated with LPNPs in a ratio of 7.5:1.0. After 24 h, cells were seeded in methylcellulose gels and allowed to grow into colonies for 14 days (n = 3). (A,C) Colonies were visualized under a microscope after MTT staining. (B,D) Colony counts with the treated ALL cells (normalized to untreated cells). Data are shown as the mean + SD. Significances were compared with CsiRNA and analyzed by two-way ANOVA (Sidak’s multiple comparisons test). ***p < 0.0002, **p < 0.002, and *p < 0.03. UT, untreated cells.

PEI-C/siSTAT5A Induces STAT5A Knockdown to Suppress ALL Tumor Growth In Vivo with an Acceptable Safety Profile

Through a series of in vitro studies above, PEI-C was considered superior to the other two LPNP formulations in terms of siRNA delivery efficiency, gene silencing capabilities, and inhibition of ALL cell growth. We then evaluated the efficacy of PEI-C in a preclinical model as well as in primary cells derived from ALL patients. A rodent xenograft model was established by subcutaneously injecting ALL-derived RS4;11 cells into triple-immunodeficient NCG mice. Five doses of CsiRNA and siSTAT5A complexes were administered at 1 mg/kg treatment, and changes in tumor volumes were assessed externally. We found a significant reduction in the tumor volume in mice injected with PEI-C/siSTAT5A complexes as compared to the PEI-C/CsiRNA treatment group (Figure 7B), without affecting the body weight (Figure 7C). The RT-qPCR analysis on excised tumor tissues indicated that PEI-C/siSTAT5A significantly suppressed the STAT5A expression compared to the PEI-C/CsiRNA treatment (Figure 7D).

A subsequent study investigated the effect of SQ administration of PEI-C/CsiRNA complexes in normal (Balb/c) mice on the blood chemistry and cell composition. We used the complexes with CsiRNA with no specific target in order to avoid potential off-target effects. Examination of complete blood counts revealed that mice treated with complexes showed no significant changes in the number of red blood cells, reticulocytes, white blood cells, and platelets compared to sham-treated mice, indicating that PEI-C complexes are generally well-tolerated (Figure 8A). The hemoglobin levels were also similar in both groups. Serum analysis indicated no significant toxicity in the mice following the administration of PEI-C complexes compared with the sham treatment (Figure 8B). Biomarker levels for liver and kidney functions, including albumin and globulin, were similar in both sham and complex-treated mice. There was no hepatic toxicity, as evidenced by no substantial differences in the levels of liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) and similar bilirubin levels in both groups. Similar levels of blood urea nitrogen (BUN) suggested unaffected kidney functions, and comparable creatine kinase levels indicated no signs of muscle-associated toxicity. The blood cell counts and serum profile results together suggest that the PEI-C/siRNA formulation is well-tolerated in vivo.

Figure 8.

Figure 8

Biocompatibility of PEI-C/siRNA complexes in a healthy BALB/c mouse. (A) Blood cell counts 24 h following a single SQ treatment with sham or PEI-C/CsiRNA complexes (n = 5). (B) Serum biochemistry profile 24 h after 5 SQ injections with sham or PEI-C/CsiRNA complexes (n = 5). Significance was determined using a parametric paired t-test. Data are presented as the mean + SD. RBC, red blood cell; WBC, white blood cell; AST, aspartate aminotransferase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; BUN, blood urea nitrogen.

PEI-C/siSTAT5A Downregulates STAT5A mRNA Expression in Primary Patient Samples

Next, the effectiveness of PEI-C for STAT5A silencing was investigated in primary cells, particularly exhibiting the Philadelphia chromosome (Ph)-like phenotype. The MN cells (from bone marrow aspirates) from 3 patients were subjected to transfection with PEI-C/CsiRNA and PEI-C/siSTAT5A complexes followed by RT-qPCR to measure the expression of STAT5A. PEI-C proved to be efficacious in delivering siSTAT5A, leading to a significant reduction in the level of STAT5A expression within these primary cells (Figure 9).

Figure 9.

Figure 9

Effects of PEI-C/siSTAT5A complexes on primary patient samples. Primary cells from 3 ALL patients were transfected with PEI-C complexes of CsiRNA and siSTAT5A (n = 2). After 3 days, RT-qPCR analysis was performed to measure the STAT5A knockdown, and results are summarized as the change in individual patients (left) and the average change among the 3 patients (right). Significance was determined by using a parametric paired t-test. Data are presented as means + SD (*p = 0.0350).

Discussion

At the forefront of siRNA-based therapeutic development, primary cells, particularly lymphocytes, are considered as less responsive to regular transfection methods. One of the complications is the slower proliferation rates of these cells, when compared with immortalized cell lines.40,41 This issue in transfection efficiency is further complicated by increased sensitivity of primary cells to the vectors used to deliver the siRNAs, which induce unwanted immune responses or cause direct cytotoxic effects.4246 However, a safe and efficient method for the siRNA transfection approach in lymphocytes and primary human PBMCs is rare. Thus, it is crucial to develop an siRNA carrier that minimizes unwanted toxicity while achieving effective transfection. Hence, we developed a library of lipopolymers designed to deliver therapeutic siRNA into lymphocytes safely and effectively. Through a comprehensive screening of our library (unpublished data), we identified three promising lipopolymers: PEI-A, PEI-B, and PEI-C, for further studies reported in this manuscript. We used these lipopolymers to prepare complexes with siRNA. We found complete binding of siRNAs with our lipopolymers at low BC50 (the w/w ratio at which 50% of siRNA binds to lipopolymers), with respective values of 0.38 for PEI-A, 0.29 for PEI-B, and 0.34 for PEI-C, signifying a high affinity between the siRNA and nanocarriers. This interaction is crucial for the formation of stable nanoparticles that protect siRNA molecules against enzymatic degradation, enhance their uptake by cells, and maximize the gene silencing efficacy.47 We were also able to pack our LPNPs of ∼200 nM (with PEI-A displaying a higher size). This was a favorable outcome that enabled us to pave the way for advancing to further studies because nanoparticles with a size range of 100–200 nm are well-suited for delivering therapeutic siRNA into living systems, as their properties enable efficient therapeutic nucleic acid transport while overcoming challenges associated with targeted delivery.48,49

Our subsequent focus was on evaluating the immune response against these lipopolymers by measuring cytokine release from different PBMCs treated with the siRNA complexes. In contrast to the high cytokine response elicited by PMA+IO stimulated cells, our LPNPs showed minimal proinflammatory cytokine release, indicating their safety upon exposure to blood components, making them particularly suited for therapeutic use in ALL. Some cytokine secretion was noted with the siRNA complexes, but the level of response was similar to siRNA alone, so that complexation with the polymers did not add additional effects in this regard. The carriers used in this study were derived from 1.2 kDa PEI and literature studies with small-molecular-weight PEI (<2 kDa), in both its original and lipid-modified forms, suggesting no or minimal cytokine induction, while higher-molecular-weight PEI (22 kDa) has been shown to elevate proinflammatory cytokines to multiple folds.44 Additional tests on their gene silencing efficacy in hard-to-transfect PBMCs demonstrated the lipopolymers’ capability to silence the target gene effectively. We found differences in response to LPNP treatments in PBMCs from HVs, which can be attributed to the inherent biological variability among individuals. Genetic variability among primary cells can impact the expression of target mRNAs, thereby affecting the siRNA-mediated silencing capability. Polymorphisms in the target gene may also hinder the effectiveness of siRNA. Variations in the expression levels of siRNA machinery components, such as the dicer, can influence siRNA effectiveness.50 In addition, siRNA can trigger immune responses that vary between patients due to individual immune system differences and epigenetic factors, potentially affecting overall silencing efficiency.51 These factors might explain the variations in the silencing efficiencies observed in different HV samples. Despite these differences and with successful transfection evidence, we proceeded to evaluate these lipopolymers in both in vitro (including immortalized and primary cells) and in vivo models of ALL. We note that such variations have an important impact on the clinical performance of siRNA therapies, but probing the underlying reasons for such differences will be left to another study.

ALL is recognized as a genetically diverse disease, characterized by a variety of genetic alterations that differ from patient to patient. An example includes the SUP-B15 cell line, derived from the bone marrow of a 9-year-old boy experiencing a second relapse, which has the ALL variant (m-bcr) of the BCR::ABL1 fusion gene.52 Conversely, the RS4;11 cell line, originating from the bone marrow of a 32-year-old woman in her first relapse, carries the KMT2A::AFF1 fusion gene.53 Our lipopolymer system complexed with siSTAT5A demonstrated a significant ability to inhibit colony formation across both cell lines. This effectiveness is independent of patient age, sex, and genetic variations underlying the disease, highlighting the promise of our lipopolymer system as a solid basis for crafting innovative therapies for ALL. Despite all three lipopolymers demonstrating promising siRNA delivery capabilities, we wanted to explore the single most effective lipopolymer for preclinical studies and found PEI-C to outperform the others. Consistent with our in vitro observations, our previous study also demonstrated that PEI-C/siSTAT5A complexes effectively silenced the STAT5A in 4 out of 8 ALL patient-derived cells, accompanied by a significant reduction in colony formation capabilities in three of the eight, reflecting a significant decrease in the growth potential of the leukemia cells.16 Based on these findings, we proceeded with PEI-C in subsequent animal studies.

The translation of siRNA therapies from in vitro to in vivo models presents considerable challenges due to the complexities of biological systems that are not encountered in controlled laboratory environments, including rapid degradation of siRNA molecules in vivo, clearance from the systemic circulation, and restricted biodistribution.54 A review of the literature on nonviral siRNA delivery methods aimed at suppressing ALL growth highlights the employment of several nonviral vectors, including HiPerFect,5557 OligoFectamine,58 Turbofect,59 and Lipofectamine 2000,6062 as well as mechanical approaches such as electroporation.6369 Notably, only two of these studies employed animal models to test the delivery efficiency. In one study, the SEM cell line of ALL was electroporated with siRNA targeting the KMT2A::AFF1 fusion gene at a concentration of 500 nM before transplantation into SCID mice, resulting in a marked improvement in survival rates.70 The use of such a high siRNA concentration underscores the challenge posed by electroporation, which is impractical for therapeutic development due to its technical issues.44 In another study, siRNAs targeting the TCF3::PBX1 fusion gene were encapsulated within lipid nanoparticles (LNPs) and administered intraperitoneally at a dosage of 2.5 mg/kg across 10 injections in an systemic ALL xenograft model, achieving a reduction in ALL cell engraftment compared to the control group.71 However, the complexity of LNP preparation, requiring specialized equipment and expertise, limits its applicability. Furthermore, the effectiveness of LNP-delivered siRNA is significantly decreased, with ∼70% of the internalized siRNA being recycled through endocytic pathways,72 which also poses a risk of inducing immunogenic reactions.73 In contrast, our research utilizing the PEI-C/siRNA complex for delivery, which simplifies the preparation process to simple mixing and incubation at room temperature without the need for specialized equipment, demonstrated significant tumor volume reduction with only 5 subcutaneous injections at a dose of 1 mg/kg siRNA, as opposed to the higher dosage of 2.5 mg/kg used in the comparative study. Our findings indicated no significant difference in body weight between the control and treatment groups, suggesting the safety of our LPNP delivery system. In addition to its in vivo inhibition of STAT5A, PEI-C also markedly reduced STAT5A levels in primary cells from three patients with a high-risk form of ALL, known as (Ph)-like positive/BCR::ABL1-like ALL. Our in vivo safety study with PEI-C/siRNA complexes revealed no significant differences in blood biochemistry parameters between the control group and the treatment group, highlighting the biocompatibility of these complexes and supporting the potential of PEI-C as a candidate for the development of siRNA-based treatments for ALL.

One limitation of our study is related to the use of local RS4;11 xenografts rather than establishing a systemic xenograft model in our preclinical model, which would have offered an advantageous scenario for reaching cells within the bone marrow. Here, we have investigated only the SQ route for therapeutic administration. However, exploring alternative routes for PEI-C/siSTAT5A (such as intravenous or intraperitoneal) may significantly enhance the biodistribution of siRNA and improve the therapeutic efficacy. In a study involving similar lipopolymers complexed with FLT3 siRNA injected via the intravenous route in an acute myeloid leukemia xenograft model, we observed increased siRNA biodistribution within the tumor samples.54 Likewise, the intravenous administration of the LPNPswith BCR::ABL1 siRNA in another chronic myeloid leukemia study resulted in a marked reduction in the tumor volume.74 Additionally, integrating patient-derived xenograft models alongside our cell-line-derived xenograft model for evaluating the therapeutic efficacy of PEI-C/siSTAT5A could provide critical insights for moving our findings toward clinical studies.

Conclusions

In this study, we introduced an LPNP system designed as a delivery platform for siRNA targeting an oncogene for ALL treatment. Here, we demonstrated enhanced specific silencing of STAT5A across different ALL cell lines with varying genetic profiles in vitro, suggesting their potential for targeting other aberrant oncogenes in other forms of ALL, such as MLL-rearranged ALL. This study marks the initial demonstration of the preclinical proof of concept, as we effectively delivered lipopolymer-mediated therapeutic siSTAT5A into a B-cell ALL xenograft model, leading to STAT5A knockdown and significant reductions in tumor growth. Our safety data confirm the biocompatibility of LPNPs, as shown by minimal induction of proinflammatory cytokines and without effects on blood cell counts and serum profiles. Exploring combination strategies involving chemotherapeutic agents with siSTAT5A LPNPs should be further considered to potentially augment the effectiveness of the existing ALL treatments.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00336.

  • (Table S1) siRNA sequences; (Table S2) primer sequences for human β-actin, STAT5A, and proinflammatory cytokines; (Table S3) sample study design for LPNP preparation; (Table S4) summary of statistical analysis for STAT5A knockdown efficiency in PBMCs by lipopolymer formulations; (Figure S1) simple siRNA transfection protocol with PEI-based lipopolymers; (Figure S2) LPNP characterization; (Figure S3) early apoptotic effects of siSTAT5A LPNPs on ALL cells in vitro (PDF)

Author Contributions

M.N. performed review and editing of the manuscript, original draft preparation, visualization, methodologies, investigation, formal analysis, data curation, and conceptualization. R.K.C., K.N., K.P., D.N.M.S., and A.P.R. performed methodologies. C.K. performed resources acquisition. X.J. and J.B. performed validation. H.U. performed review and editing of the manuscript, supervision, project administration, funding acquisition, formal analysis, and conceptualization.

The work was supported by funding from Canadian Institutes of Health Research (CIHR) via a Project Grant, the Natural Sciences and Engineering Council of Canada (NSERC) via a Discovery Grant, and Alex’s Lemonade Stand Foundation (ALSF) via an Innovation Grant. Additional support was provided by the RJH Biosciences, Inc. via a MITACS Accelerate Fellowship to M.N., A.P.R., and D.N.M.S. as trainees at the University of Alberta. M.N. is supported by the Rachel Mandel Scholarship in Lymphoma and Other Blood Cancers through his graduate student scholarships. Equipment support was provided by the Edmonton Civic Employees–Charitable Assistance Fund.

All animal experiments were performed in accordance with procedures preapproved by the Health Sciences Laboratory Animal Services (HSLAS), University of Alberta. The blood donations for the isolation of mononuclear cells were obtained from Canadian Blood Services (Vancouver, BC) under ethics approval and without donor information. The ALL cells were obtained from Children’s Oncology Group (COG; Philadelphia, PA) under an approved ethics protocol (Protocol No. AALL20B1-Q).

The authors declare the following competing financial interest(s): M.N., A.P.R., and D.N.M.S. were supported by a fellowship from RJH Biosciences Inc. R.K.C. and H.U. have ownership interest in RJH Biosciences Inc. (Edmonton, Alberta, Canada).

Supplementary Material

pt4c00336_si_001.pdf (303.6KB, pdf)

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