Tailored Nanoparticle Codelivery of antimiR-21 and antimiR-10b

Aug 10, 2016 - Considering the continued abysmal clinical prognosis of glioblastoma, its general chemoresistance to standard treatment using Temozolom...
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Tailored Nanoparticle Codelivery of antimiR-21 and antimiR-10b Augments Glioblastoma Cell Kill by Temozolomide: Toward a “Personalized” Anti-microRNA Therapy Jeyarama S. Ananta, Ramasamy Paulmurugan,* and Tarik F. Massoud* Laboratory of Experimental and Molecular Neuroimaging, Molecular Imaging Program at Stanford (MIPS), and Bio-X Program, Stanford University School of Medicine, Stanford, California 94305-5427, United States ABSTRACT: Glioblastoma remains an aggressive brain malignancy with poor prognosis despite advances in multimodal therapy that include standard use of Temozolomide. MicroRNA-21 (miR-21) and microRNA10b (miR-10b) are oncomiRs overexpressed in glioblastoma, promoting many aspects of cancer biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 (antimiR-21) and antisense miR-10b (antimiR10b) might beneficially knockdown endogenous miR-21 and miR-10b function and reprogram cells prior to Temozolomide treatment. PLGA nanoparticles were effective in intracellular delivery of encapsulated oligonucleotides. Concentrations of delivered antimiR-21 and antimiR10b were optimized and specifically tailored to copy numbers of intracellular endogenous microRNAs. Coinhibition of miR-21 and miR10b significantly reduced the number of viable cells (by 24%; p < 0.01) and increased (2.9-fold) cell cycle arrest at G2/M phase upon Temozolomide treatment in U87 MG cells. Cell-tailored nanoparticle-assisted concurrent silencing of miR-21 and miR-10b prior to Temozolomide treatment is an effective molecular therapeutic strategy in cell culture, warranting the need for further studies prior to future in vivo “personalized” medicine applications. KEYWORDS: glioblastoma, PLGA nanoparticle, microRNA-21, microRNA-10b, antisense miR-10b, antisense miR-21, anti-microRNA therapy, personalized medicine

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INTRODUCTION Brain glioblastoma is an aggressive, recalcitrant malignancy with high morbidity and mortality.1 Despite multimodal treatments that include surgical resection, radiotherapy, and adjuvant chemotherapy using Temozolomide, glioblastoma remains a disease with poor prognosis.2,3 Hence, there is a need to develop more effective molecular therapeutic strategies targeting aspects of glioma biology. Dysregulated microRNA expression has been associated with oncogenesis of many cancers, including glioblastoma, thus affecting proliferation, apoptosis, invasion, migration, and resistance to therapy.4−8 Oncogenic microRNAs (oncomiRs) promote carcinogenesis by targeting cell cycle regulators and pro-apoptotic genes. Inhibition of endogenous oncomiRs using sequence specific antisense oligonucleotides is a promising molecularly targeted anticancer therapeutic approach for glioblastoma that requires further evaluation and optimization prior to future clinical translation. Microarray based profiling of microRNA expression in glioblastomas has identified microRNA-21 (miR-21) and microRNA-10b (miR-10b) as oncomiRs that are significantly overexpressed in glioblastoma.5,9,10 Indeed, miR-21 was one of the first oncomiRs studied in various cancers including glioblastoma, controlling its tumor invasion, apoptosis, cell proliferation, and chemoresistance.9,11−15 miR-10b is another © XXXX American Chemical Society

microRNA highly expressed in metastatic breast cancer, pancreatic adenocarcinomas, and glioblastoma.5,10,16,17 It is also an oncogenic microRNA expressed specifically in glial tumors but not in normal brain cells.10 miR-10b inhibition has been shown to compromise cell proliferation, survival, migration, and invasion.10,18 Recently, coinhibition of miR-21 and miR-10b has been shown to induce cell cycle arrest and reduce migration and apoptosis in glioblastoma cells.19 The exact mechanisms controlling these changes are not yet understood completely. Considering the continued abysmal clinical prognosis of glioblastoma, its general chemoresistance to standard treatment using Temozolomide, and the significant role of microRNAs in cancer cell biology, we hypothesized that antisense oligonucleotide-assisted downregulation of miR-21 and miR-10b function in glioblastoma cells might enhance their therapeutic response to Temozolomide. Compared to endogenous microRNAs, synthetic naked microRNAs rapidly degrade in plasma.20 Despite improvements in their stability by structural modification, therapeutic antisense microRNAs require efficient encapsulation systems Received: May 2, 2016 Revised: July 6, 2016 Accepted: August 10, 2016

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DOI: 10.1021/acs.molpharmaceut.6b00388 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics to achieve adequate delivery to intended targets.21 Commercially available transfection agents, such as Oligofectamine, form stable polyamino lipoplexes with sense and antisense microRNAs to deliver them across cell membranes. However, such agents are not suitable for in vivo applications owing to systemic toxicity, degradation of microRNAs by serum nucleases, offtarget effects, and rapid renal clearance.22 Herein, we investigate the use of poly[lactic-co-glycolic]acid (PLGA) nanoparticles to encapsulate and deliver antisense miR-21 (antimiR-21) and antisense miR-10b (antimiR-10b) to glioblastoma cells in culture. Recently, we and others have shown that PLGA nanoparticles are effective intracellular carriers for antimiR-21, offering sustained release and efficient downregulation of endogenous miR-21 compared to commercial transfection agents.23−25 In this study, we use PLGA nanoparticles as nanocarriers for concurrent delivery of antimiR-21 and antimiR-10b and investigate the effects of PLGA nanoparticle-assisted coinhibition of miR-21 plus miR-10b on Temozolomide treatment, a strategy that has not been explored in glioblastoma cells previously. Importantly, we deliver antimiR-21 and antimiR10b concentrations specifically tailored to the intracellular copy numbers of target endogenous microRNAs in the cells being treated and argue that this novel approach is a rational and effective mode of inducing concurrent endogenous miR-21 and miR-10b downregulation in glioblastoma cells, in a manner that could be clinically translatable to “personalized” medicine applications in the future. The resultant miR-21 and miR-10b suppression prior to Temozolomide treatment significantly reduces the viability of cells and increases cell cycle arrest in G2/M phase for Temozolomide-treated cells. Moreover, coinhibition of miR-21 and miR-10b increases phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), and Homeobox 10 (HOXD10) expression. Our results demonstrate that intracellular delivery of PLGA nanoparticles encapsulating antimiR-21 and antimiR-10b is effective in reducing cell proliferation and increases chemosensitivity of glioblastoma cells to Temozolomide.

washed them twice with DNase/RNase free water to remove nonencapsulated antimiR-21 (or antimiR-10b) and excess PVA. We passed the washed particles through a 0.45 μm filter to remove any large aggregates. We freeze-dried the particles with trehalose (1.5% w/w) as a cryopreservant for long-term storage. For quantitation of nanoparticle uptake by glioblastoma cells, we prepared sense miR-542-3p (UpGpUpGACAGAUUGAUAACUGpApApA) loaded PLGA nanoparticles using the same procedure as above. We measured the nanoparticle hydrodynamic diameter, size distribution, and surface charge (ζ-potential) using a dynamic light scattering (DLS) technique (Zetasizer Nano ZS, Malvern Instruments, UK). We measured the encapsulation efficiency of antimiR-21 (or antimiR-10b or miR-542-3p) loaded PLGA nanoparticles using an organic/aqueous extraction method. We quantified the amount of antimiR-21 (or antimiR-10b) present in the aqueous fraction using the Quant-iT RNA-quantitation kit (Invitrogen, Carlsbad, CA), according to the manufacturer’s guidelines. We calculated the “encapsulation efficiency” using the following formula: encapsulation efficiency(%) ⎛ amount of antisense miRNA quantified in NPs ⎞ ⎟ =⎜ ⎠ ⎝ total amount of antisense miRNA used × 100

Cell Culture. We purchased the human glioblastoma cell lines, U87 MG (HTB-14), LN229 (CRL-2611), and T98G (CRL-1690), as well as cortical neuronal brain cells HCN-2 (CRL-10742) from American Type Culture Collection (ATCC; VA). We maintained the cells in Eagle’s minimum essential medium (MEM; Corning Cellgro; VA) supplemented with 10% fetal bovine serum (FBS) and penicillin− streptomycin (100 U/mL) and incubated at 37 °C in a fully humidified atmosphere with 5% CO2. We routinely passaged these cells at two to three day intervals and used cells of less than 40 passages for all our experiments. PLGA Nanoparticle Assisted Antisense microRNA Transfection of Glioblastoma Cells. For PLGA nanoparticle assisted microRNA transfection studies, we compared four different treatment conditions (nontransfected control, antimiR-21 transfection alone, antimiR-10b transfection alone, and antimiR-21 plus antimiR-10b cotransfection). We incubated the U87 MG cells with PLGA nanoparticles encapsulating various concentrations of antisense microRNAs (0−25 pmoles) (polymer concentration: 0−20 μg of PLGA) in reduced serum (2%) medium for 24 h at 37 °C and 5% CO2. For quantitation of nanoparticle uptake by glioblastoma cells, we incubated U87 MG cells with biotinylated miR-542-3p encapsulated PLGA nanoparticles at various cell-to-nanoparticle ratios (from 1:0 to 1:10,000) for 24 h at 37 °C and 5% CO2. MicroRNA Extraction and Quantitative Real-Time PCR. We isolated the total RNA (mRNA and microRNA) using a mirVana RNA extraction Kit (Life Technologies, CA) using the manufacturer’s protocol. For microRNA quantitation using PCR, we reverse transcribed 15 ng of total RNA using RT-primers of miR-21 and miR-10b using TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, United States). We performed real-time PCR using 5 μL of cDNA (750 pg of RNA equivalent) combined with TaqMan real time PCR reagents of miR-21 or miR-10b. We performed

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METHODS Synthesis and Physicochemical Characterization of Antisense microRNA Loaded PLGA Nanoparticles. We used phosphorothioate modified antimiR-21 oligonucleotide with the following sequence UpCpApACAUCAGUCUGAUAAGpCpUpA and antimiR-10b oligonucleotide with the sequence CpApCpAAAUUCGGUUCUACAGGpGpUpA for our endogenous microRNA target blocking studies. The oligonucleotides were custom synthesized in the PAN facility at Stanford University with >90% purity. We prepared PLGA nanoparticles encapsulating antimiR-21 and antimiR-10b using a double emulsion solvent evaporation technique reported previously.23 Briefly, we complexed antimiR-21 (10 nmol) or antimiR-10b (10 nmol) with spermidine at room temperature for 15 min in DNase/RNase free water. We added dropwise the antimiR-21 (or antimiR-10b) and spermidine complex to a solution of PLGA (20 mg) in dichloromethane (1 mL) while stirring. We then probe-sonicated the solution for 60 s at 40% amplitude in an ice bath. We further emulsified the first emulsion thus formed by sonication (60 s at 60% amplitude) in 2% poly(vinyl alcohol) (PVA) solution and stirred the emulsion continuously under reduced pressure to evaporate the organic solvent. We collected the resulting nanoparticles by centrifugation (100 K MWCO membrane; 5000 g; 30 min) and B

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Figure 1. miR-21 and miR-10b expression analysis by Taqman-based RT-PCR. Endogenous levels of miR-21 (a) and miR-10b (b) expression in different glioblastoma cells (U87 MG, LN229, and T98G) and control cortical neuronal (HCN-2) cells (results are presented as mean ± standard deviation; ** indicates p ≤ 0.01).

all reactions in a 20 μL volume. PCR parameters consisted of 2 min incubation at 50 °C, followed by activation of the DNA polymerase at 95 °C for 10 min and 60 cycles of 95 °C × 15 s, 60 °C × 60 s in an Eppendorf real-time PCR system. We normalized the miR-21 or miR-10b expression to RNU66. We used a single factor ANOVA method for determining statistical significance and considered a p value of ≤0.05 as significant. For precise quantitation of total number of copies of miR-21 and miR-10b in glioblastoma cells, we reverse transcribed the total RNA from a known number of cells (90,000−100,000). We quantified the number of copies of endogenous miR-21 and endogenous miR-10b per cell using a standard curve developed with known number of copies of miR-21 and miR-10b (custom synthesized at Stanford University). For quantitation of nanoparticle uptake experiments, we isolated the total RNA using a mirVana RNA extraction Kit. To pull down the biotinylated-miR-542-3p released from PLGA nanoparticles, we further incubated the isolated total RNA with 10 μL of Streptavidin-coupled Dynabeads (Life Technologies AS, Norway) for 2 h. We washed the miR-542-3p bound to Dynabeads three times with PBS using a magnetic stand. We reverse transcribed the Dynabeads bound to miR-542-3p using TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, United States) and performed real-time PCR using 5 μL of cDNA combined with TaqMan real time PCR reagents. Cell Viability Studies. We used an MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay to evaluate the cell proliferation and cytotoxicity of Temozolomide on antisense microRNA transfected and nontransfected glioblastoma (U87 MG, LN229) cells. We seeded the cells in a 96-well plate at 5 × 103 cells/well density. We incubated the cells with antisense microRNAs for 24 h at 37 °C and 5% CO2. We exchanged the medium for 100 μL of fresh medium (2% FBS) supplemented with Temozolomide in concentrations ranging from 0 to 500 μM. We then incubated the cells for a further 48 h. At 48 h post Temozolomide exposure, we aspirated the cell culture medium and incubated the cells with 100 μL of medium (containing 12 mM MTT) for 3 h at 37 °C in 5% CO2. We then carefully removed the medium and dissolved the reduced formazan crystals in 100 μL of DMSO by incubating at 37 °C for 15 min in the dark. Next, we calculated cell viability by measuring the absorbance (Abs) at 540 nm using a microplate reader (Infinite M1000 Pro,

Tecan Group, Switzerland) and comparing with the control cells. The relative cell viability (%) compared to control cells was calculated as follows: ⎡ Abs(sample) − Abs(blank) ⎤ cell viability(%) = ⎢ ⎥ × 100 ⎣ Abs(control) − Abs(blank) ⎦

We used a single factor ANOVA method to determine statistical significance and considered a p value of ≤0.05 as significant. Cell Cycle Analysis. For cell cycle distribution studies, we harvested the transfected and control U87 MG cells after the specified experimental conditions and washed them with PBS. We then fixed the cells in ice cold 70% ethanol and stained them with 0.5 mL of PBS containing 0.5 μg/mL propidium iodide, 10 μg/mL RNase A, and 0.1% TritonX-100. We incubated the cells for 30 min at room temperature in the dark and performed the analysis using a FACS Aria III (BD Biosciences, CA) cell sorter. We analyzed the data using FlowJo FACS analysis software (Tree Star, OR). Western Blot Analysis. For Western blot analysis, we washed the cells with PBS after the experimental conditions to remove traces of culture medium and debris and lysed the cells in RIPA buffer (Pierce Biotechnology, IL). We estimated the protein content of the supernatant solution using a Pierce 660 protein assay kit (Life Technologies, CA). We then resolved 10 μg of protein in 4−12% gradient SDS/PAGE (Invitrogen) and electroblotted onto a 0.2 μm pore size nitrocellulose membrane (Schleicher and Schuell Biosciences, GmbH). We used Colorplus (New England Biolabs, MA) prestained protein marker to confirm the molecular mass and complete transfer of protein to the membrane. Upon transfer to the membrane we incubated the membrane in the blocking solution (5% nonfat dry milk in TBS-T [TBS with 0.1% Tween 20] buffer) with respective antibody (rabbit mAb PTEN, rabbit mAb PDCD4, rabbit Ab HOXD10, rabbit mAb Caspase-3; Cell Signaling Technology, MA) at the manufacturer’s suggested dilution, overnight and at 4 °C on a rotating platform. We washed the membrane three times with TBS-T and incubated with HRPconjugated antirabbit secondary antibody for 2 h at room temperature. We washed the membrane a further three times with TBS-T buffer before incubation with the chemiluminescent HRP substrate LumiGlo (Cell Signaling, MA), following the manufacturer’s instructions. We stripped the same membrane and reprobed with rabbit mAb GAPDH (Cell C

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Figure 2. Absolute copy number quantification for the expression of miR-21 and miR-10b in glioblastoma cells by Taqman-based RT-PCR. Number of copies of miR-21 (a) and miR-10b (b) expressed in different glioblastoma cells (U87 MG, LN229, and T98G).

Figure 3. Physicochemical characterization of antimiR-21 and antimiR-21 loaded PLGA nanoparticles. (a) Schematic representation of PLGA nanoparticles encapsulating antisense miRNA molecules. (b) Representative DLS distribution of antisense miRNA loaded PLGA nanoparticles. (c) Representative ζ-potential graph of antisense miRNA loaded PLGA nanoparticles. (d) Table showing the size, surface charge, polydispersity index, and encapsulation efficiency of antisense miRNA loaded PLGA nanoparticles.

Signaling Technology, MA) to control for protein loading. We then detected the chemiluminescense signal indicating protein levels using an IVIS optical CCD camera (Caliper, CA).

significantly overexpressed miR-21 and miR-10b (Figure 1). U87 MG cells overexpressed miR-21 by 10.5 ± 0.4-fold (p < 0.01), and LN229 cells overexpressed by 3.1 ± 0.01-fold (p < 0.01). We observed no statistically significant overexpression of miR-21 in T98G cells (p > 0.05) (Figure 1a). Similarly, U87 MG ((4 ± 0.03) × 109) and LN229 cells ((4.6 ± 0.16) × 109) had 109-fold overexpression of miR-10b (p < 0.01), and T98G cells had (1.8 ± 0.13) × 1010-fold (p < 0.01) overexpression of miR-10b (Figure 1b). We also quantified the number of copies per cell for miR-21 and miR-10b in each of the glioblastoma cell lines (U87 MG, LN229, T98G) using qRT-PCR (Figure

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RESULTS Differential Expression of miR-21 and miR-10b in Glioblastoma Cells. We evaluated miR-21 and miR-10b expression levels in U87 MG, LN229, and T98G cells, as well as HCN-2 cells. We performed Taqman-based quantitative RTPCR (qRT-PCR) analysis for microRNA expression levels. Compared to control HCN-2 cells, glioblastoma cells D

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Figure 4. Antisense miRNA release profile from PLGA-nanoparticles. (a) antimiR-21 and (b) antimiR-10b release from PLGA nanoparticles at pH 7.4, 37 °C, and 5% CO2 (results are presented as mean ± standard deviation).

Figure 5. PLGA mediated antisense miRNA transfection in U87 MG cells. (a) Percentage uptake of miR-542-3p nanoparticles by U87 MG cells at different incubation concentration. (b) PLGA nanoparticle assisted endogenous miR-21 suppression using different concentrations of antimiR-21. (c) Simultaneous downregulation of endogenous miR-21 and miR-10b functions in U87 MG cells cotreated with PLGA nanoparticles encapsulated with antimiR-21 and antimiR-10b (results are presented as mean ± standard deviation; ** represents p ≤ 0.01).

2). We observed (60 ± 1.4) × 103 copies of endogenous miR21 in U87 MG cells. LN229 ((23 ± 1.5) × 103 copies/cell) and T98G ((25 ± 0.6) × 103 copies/cell) also expressed significantly higher numbers of miR-21 copies per cell (Figure 2a). Compared to the number of copies of miR-21 in glioblastoma cells, we observed relatively lower numbers of endogenous miR-10b copies in glioblastoma cells (Figure 2b). U87 MG cells expressed 330 ± 10 copies, LN229 cells had 500 ± 8 copies, and T98G had 1300 ± 43 copies of miR-10b/cell. Thus, miR-21 and miR-10b are oncomiRs significantly overexpressed in three glioblastoma cell lines (U87 MG, LN229, T98G), and glioblastoma cells express higher copy numbers of endogenous miR-21 compared to miR-10b. PLGA Nanoparticle-Delivered antimiR-21 and antimiR-10b Silence Respective Endogenous microRNA Functions in U87 MG Cells. We evaluated PLGA nanoparticles that carry antisense microRNAs as transfection agents for simultaneous suppression of endogenous miR-21 and miR-

10b functions in U87 MG cells. The nanoparticles in our study were spherical in shape with a hydrodynamic diameter of ∼175 nm. They had a net negative surface charge (∼ −29 mV) and exhibited high encapsulation efficiency (>75%) for both antimiR-21 and antimiR-10b (Figure 3). Based on the spherical shape and diameter of the nanoparticles, we calculated the number of nanoparticles per mg of the polymer. Using the concentration of encapsulated antisense microRNA per mg of the polymer, we then estimated that an antimiR-21 loaded PLGA nanoparticle had about 970 antimiR-21 molecules per particle, and antimiR-10b loaded PLGA nanoparticles encapsulated about 990 antimiR-10b molecules per particle. Next, we performed release studies on antisense microRNA encapsulated PLGA nanoparticles at physiological conditions (pH 7.4; 37 °C and 5% CO2) (Figure 4). We quantified the number of antisense microRNAs released from nanoparticles over time by qRT-PCR. We used a standard curve generated from a known number of copies of antimiR-10b and antimiRE

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Figure 6. Antiproliferative and cell viability studies on antimiR-21, antimiR-10b, and TMZ cotreated glioblastoma cells. (a) Phase contrast microscopic images of control and antisense miRNA transfected cells treated with 250 μM TMZ. (b) In vitro cell viability MTT assays on control and antisense miRNA transfected U87 MG cells treated with different concentrations of TMZ (results are presented as mean ± standard deviation; * represents p ≤ 0.05, ** represents p ≤ 0.01).

uptake of nanoparticles (miR-542-3p copy numbers equivalent to 1−2.5% of incubated particles) at all concentrations of nanoparticles studied (Figure 5a). Though the percentage uptake of nanoparticles remained low at all incubation concentrations, importantly, the absolute number of nanoparticles internalized by U87 MG cells increased with higher nanoparticle incubation concentrations (Figure 5a). We subsequently incubated U87 MG cells with higher concentrations (0−25 pmol; 0−20 μg of PLGA) of antimiR-21 encapsulated PLGA nanoparticles and analyzed the downregulation of endogenous miR-21 using Taqman-based qRTPCR (Figure 5b). At 48 h after transfection, we observed significant (70%) downregulation of endogenous miR-21 in U87 MG cells by antimiR-21 delivered by PLGA nanoparticles (Figure 5b). For PLGA nanoparticle assisted coinhibition of miR-21 and miR-10b in U87 MG cells, we incubated the cells with PLGA nanoparticles encapsulating antimiR-21 (6.25 pmol) and antimiR-10b (6.25 pmol). We observed significant (59%; p < 0.01) downregulation of endogenous miR-10b and miR-21 (55%; p < 0.01) when U87 MG cells were transfected concurrently with antimiR-21 and antimiR-10b loaded PLGA nanoparticles (Figure 5c). These results show that antimiR-21 and antimiR-10b were effective in specifically blocking endogenous miRNA functions and that PLGA nanoparticles encapsulating antisense microRNAs are very effective at coinhibition of miR-21 and miR-10b. Antiproliferative and Cytotoxic Effects of antimiR-21, antimiR-10b, and Temozolomide Cotreatment in Glioblastoma Cells. After confirming the simultaneous downregulation of miR-21 and miR-10b in U87 MG cells following cotransfection with antimiR-21 and antimiR-10b, we studied the effect of miR-21 and miR-10b downregulation on

21 as a reference to estimate the copy numbers. AntimiR-21 encapsulated PLGA nanoparticles exhibited a 13% release at 24 h postincubation followed by 26% release at 48 h (Figure 4a). We observed 9% release at 24 h followed by 21% release at 48 h for antimiR-10b encapsulated nanoparticles (Figure 4b). Even after 4 days of incubation, we observed that >50% of the antisense molecules remained encapsulated within PLGA nanoparticles for both antimiR-21 and antimiR-10b. Thus, the use of PLGA nanoparticles as efficient carriers of antisense oligonucleotides offers both localized high concentrations of intracellular antisense microRNAs and gradual and sustained release of encapsulated molecules over a long period. To assess the feasibility of PLGA nanoparticles delivering sufficient copy numbers of different antisense molecules for simultaneous endogenous miR-21 and miR-10b downregulation, we first investigated the percentage uptake of our PLGA nanoparticles (these were devoid of any targeting moieties to cell surface markers) by U87 MG cells relative to their incubation concentration (Figure 5a). We used miR-542-3p encapsulated PLGA nanoparticles for these internalization studies because glioblastoma cells do not endogenously express this microRNA. MiR-542-3p loaded PLGA nanoparticles exhibited similar size, surface charge, and release profile as our antimiR-21 and antimiR-10b loaded PLGA nanoparticles (data not shown). U87 MG cells were seeded in a 6-well plate and incubated with different cell-to-nanoparticle ratios (from 1:0 to 1:10,000) of miR-542-3p encapsulated PLGA nanoparticles for 24 h. The incubated cells were washed thrice with PBS to remove any extracellular nanoparticles. We quantified the nanoparticle uptake by measuring the number of intracellular copies of miR-542-3p using qRT-PCR, after using different cell-to-nanoparticle ratios. We observed a very low F

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Figure 7. (a) Flow cytometry analysis of cells stained with propidium iodide for cell cycle status. (b) Table showing the percentage cell distribution at different phases of cell cycle for control cells and antisense miRNA transfected cells upon treatment with 250 μM TMZ.

Figure 8. Immunoblot analysis and graphs showing PTEN, PDCD4, HOXD10, and GAPDH protein levels after different treatment conditions.

500 μM) (Figure 6). As PLGA nanoparticles are effective intracellular carriers for antisense molecules, offering significant and sustained downregulation of endogenous miRNA over longer therapeutic window, we hypothesized that in the present study treating glioblastoma cells with temozolamide at 24 h

antiproliferation and cell viability of Temozolomide treated glioblastoma cells. The cells were treated with nanoparticle encapsulated 6.25 pmol of antimiR-21 and 6.25 pmol of antimiR-10b, and then tested for consequent antiproliferative effects in the presence of varying doses of Temozolomide (0 to G

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when cells were transfected with antimiR-10b alone or in combination with antimiR-21 prior to Temozolomide treatment, we observed a higher level of HOXD10 expression (Figure 8).

provides the optimal time window for significant downregulation of miR-21 and miR-10b function prior to temozolamide treatment. We observed a significant reduction in the number of U87 MG cells when they were transfected with antimiR-21 plus antimiR-10b before treatment with Temozolomide (Figure 6a). We also evaluated cell viability by measuring the mitochondrial activity using an MTT assay. At all concentrations of Temozolomide studied, PLGA nanoparticle-mediated knockdown of miR-21 and miR-10b resulted in significantly decreased cell viability in U87 MG cells. The decrease in cell viability was even more significant with simultaneous miR-21 plus miR-10b functional knockdown prior to Temozolomide treatment compared to Temozolomide treatment alone (Figure 6b). At 250 μM Temozolomide, inhibition miR-21 or miR-10b alone induced a 12% reduction in cell viability (p < 0.05). However, at the same Temozolomide concentration, coinhibition of miR-10b plus miR-21 resulted in 18% (p < 0.01) decrease in the number of viable cells. At 500 μM Temozolomide, the viability decreased by 24% (p < 0.01) when U87 cells were cotransfected with antimiR-21 plus antimiR-10b. Conversely, U87 MG cells treated with Temozolomide alone resulted in only 10% decrease in viability, and Temozolomide treatment with antimiR-21 or antimiR-10b alone resulted in 14% reduction (Figure 6b). miR-21 and miR-10b Downregulation Induces Cell Cycle Arrest at G2/M Phase in U87 MG Cells. To better understand the decrease in cell viability when U87 MG cells were transfected with antimiR-21 and antimiR-10b, we performed cell cycle analysis using FACS. We did not observe any significant accumulation in G2/M phase when U87 MG cells were transfected with antimiR-10b or cotransfected with antimiR-10b plus antimiR-21 in the absence of Temozolomide treatment (Figure 7). Temozolomide treatment alone induced 26% increase in G2/M cell accumulation compared to untreated control cells. When U87 MG cells were cotransfected with antimiR-21 plus antimiR-10b prior to Temozolomide treatment, we observed nearly 2.7-fold increase in G2/M phase accumulation compared to Temozolomide treatment alone (Figure 7b). This 2.7-fold increase in cell accumulation at G2/ M phase suggests that simultaneous downregulation of miR-21 and miR-10b prior to Temozolomide treatment induces cell cycle arrest and inhibits the proliferation of U87 MG cells. miR-21 and miR-10b Downregulation Prior to Temozolomide Treatment Increases PTEN, PDCD4, and HOXD10 Expression in U87 MG Cells. To explore the effects of cotransfection of antimiR-21 and antimiR-10b prior to Temozolomide treatment on various cellular pathways, we performed immunoblot analysis for key targets of miR-21 (PTEN, PDCD4) and miR-10b (HOXD10). Figure 8 shows the immunoblot results for PTEN, PDCD4, and HOXD10. Temozolomide treatment alone on nontransfected cells induced an increased PTEN expression. However, when U87 MG cells were transfected with antimiR-21 prior to Temozolomide treatment, we observed a surge in PTEN expression. Similarly, U87 MG cells transfected with antimiR21 prior to Temozolomide treatment exhibited higher PDCD4 expression, whereas Temozolomide treatment of nontransfected and antimiR-10b transfected cells did not induce any increased expression of PDCD4. We observed a similar expression pattern for HOXD10. Temozolomide treatment alone or with antimiR-21 transfection prior to treatment did not induce any increase in HOXD10 expression. However,

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DISCUSSION PLGA Nanoparticle Delivery of Therapeutic Oligonucleotides. For therapeutic strategies targeting the brain, the presence of the blood−brain barrier (BBB) presents a significant challenge. Systemically delivered antisense microRNAs distribute broadly into most tissues except the CNS owing to the presence of BBB.21 The BBB consists of specialized endothelial cells and astrocyte endfeet that line capillaries and prevent the nonregulated delivery of bloodborne chemicals and therapeutics. Unique size, shape, and surface properties of nanoparticles make them attractive drug and gene delivery carriers to penetrate the BBB into the brain. Indeed, cationic lipid-based and poly(amido amine) dendrimer (PAMAM) based nanocarriers have been shown as efficient gene and microRNA delivery vehicles to the brain.26,27 However, a major drawback with cationic delivery vehicles is their toxicity, poor microRNA loading efficiency, and insufficient nonsustained delivery of microRNAs at the target site.22,28 Compared to most other types of delivery vehicles, PLGA nanocarriers have significantly greater advantages, such as biocompatibility, ability to encapsulate hydrophobic and hydrophilic drugs, sustained release of encapsulated molecules over time, long-term stability of encapsulated molecules, functionalizable external surface for targeting, and FDA approval for their use. We here show that PLGA nanoparticles are efficient intracellular delivery vehicles for antimiR-21 as well as antimiR-10b, allowing sustained release of both encapsulated antisense microRNAs and resulting in prolonged endogenous suppression of oncomiRs in glioblastoma cells. We had previously demonstrated similar physicochemical advantages of these nanoparticles in delivery of antimir-21 alone.23 Importantly, there is extensive research literature regarding the ability of targeted PLGA nanoparticles to cross the intact BBB.29 Overexpression of miR-21 and miR-10b in Glioblastoma Cells. Dysregulated microRNA expression is commonly reported in various human cancers. There are more than 300 microRNAs that are reported as either upregulated or downregulated in glioblastoma, and studies have shown that many of the established glioblastoma cell lines exhibit elevated miR-21 and miR-10b levels.5,9,10,30 Moreover, antisense oligonucleotide mediated knockdown of either endogenous miR-21 or miR-10b has been shown to impair oncogenic properties of cancer cells.9,11,13,14,18,19,31,32 In agreement with prior studies, we observed a significant expression of miR-21 and miR-10b in U87 MG, LN229, and T98G glioblastoma cells (Figure 1). Compared to neuronal cells (HCN-2), glioblastoma cells exhibited up to 10-fold increase in endogenous miR-21 levels (Figure 1a) and a nearly 1010-fold increase in miR-10b expression (Figure 1b). However, we contend that measuring the fold-change in the expression of endogenous oncomiRs is only a relative measurement of their quantity because the target microRNAs could be either overexpressed in glioblastoma cells compared to control cells (e.g., in the case of miR-21, this results in a spuriously lower-fold value) or not expressed in control cells (in the case of miR-10b, this manifests as a hugefold value). H

DOI: 10.1021/acs.molpharmaceut.6b00388 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics Molecular Treatment Tailored to Intracellular Levels of Endogenous microRNAs. If a desirable therapeutic goal is to concurrently silence multiple endogenous microRNAs and thus rationally achieve abrogation of multiple molecular pathways, and in turn, this resulting in safe and effective cancer cell kill, how then to deal with different expression levels of these intracellular targets? Indeed, one of the significant challenges to be addressed with RNA interference strategies is the consequence of simply flooding the cell with therapeutic oligonucleotides, resulting in unintended oversaturation of endogenous small RNA pathways, and leading to severe side effects or death. This has been demonstrated in mice treated with high doses of interfering short hairpin RNA (shRNA) molecules.33 The observed toxicity was owing to intracellular overexpression of shRNAs and not off-target effects or delivery vector induced toxicity. We here argue that analogous oversaturation of cells with delivered exogenous therapeutic microRNAs could in theory lead to unwarranted side effects, especially when using excessive concentrations of delivered antisense microRNA cocktails not specifically tailored to levels of endogenously expressed oncomiRs. To our knowledge, this concept of careful optimization of levels of therapeutic microRNAs to the exact copy numbers of endogenous microRNA targets within glioblastoma cells has not been advocated previously. This notion here presented in our study has strong translational implications for promotion of customized and personalized medicine approaches within the clinical setting, whereby, for example, future clinical treatments using microRNAs would be tailored to the exact levels of patients’ endogenous microRNA targets measured initially from surgical specimens or circulating biomarkers. As a demonstration of this principle, for antisense oligonucleotide assisted simultaneous knockdown of miR-21 and miR-10b in the glioblastoma cell lines we used, we argue that it would be important first to quantify the copy numbers of miR-21 and miR-10b in each cell type to then tailor the appropriate dose of delivered therapeutic antisense microRNA and enable safe and effective downregulation of target microRNAs. Based on our qRT-PCR method, we observed that miR-21 is expressed much more (25,000−60,000 copies/ cell) in glioblastoma cells compared to miR-10b (500−1500 copies/cell) (Figure 2a,b). These results suggest that to achieve tailored and efficient concurrent suppression of miR-21 and miR-10b it would be necessary to intracellularly deliver many more antimiR-21 molecules as compared to antimiR-10b. Considering the potentially significant biological implications of oversaturating RNA interference pathways with high doses of antisense microRNA, we hypothesized that for simultaneous downregulation of miR-21 and miR-10b in glioblastoma cells, distinct populations of PLGA nanoparticles encapsulating either antimiR-21 or antimiR-10b would offer greater advantages over coencapsulating antimiR-21 and antimiR-10b within the same nanoparticle. Indeed, coloading antimiR-21 and antimiR-10b within single nanoparticles presents considerable technical challenges such as optimizing the loading efficiency and controlling the release of individual antisense microRNAs. Moreover, coloading multiple antisense microRNAs in a single nanoparticle population would not be suitable for tailoring the number of nanoparticles required to significantly downregulate target microRNAs, considering the vast difference in copy numbers of endogenous miR-21 and miR-10b in our glioblastoma cells. Hence, for our knockdown

studies we prepared individual populations of PLGA nanoparticles encapsulating either antimiR-21 or antimiR-10b. Targeted versus Nontargeted Nanoparticles to Deliver Tailored Anti-microRNA Therapy. The sustained release of antisense molecules from our PLGA nanoparticles (Figure 4) is advantageous, as a recent study has shown that continuous, local, and targeted intratumoral infusion of antimiR-10b may be an efficient therapeutic strategy for malignant brain tumors.34 For simultaneous silencing of endogenous miR-21 and miR10b, which are expressed in significantly different amounts in glioblastoma cells, it would be imperative first to study the efficacy of these nanoparticles to intracellularly deliver and sustainedly release sufficient copies of each antisense oligonucleotide. We therefore investigated the percentage uptake of miR-542-3p loaded nanoparticles in U87 MG cells as a function of their incubation concentration using qRT-PCR (Figure 5a). Though the percentage uptake remained low at all concentrations of nanoparticles studied, the absolute number of our nontargeted nanoparticles internalized by cells increased with increasing concentrations of nanoparticles in the culture medium (Figure 5a). Of note, a similarly low percentage uptake was observed in one nanoparticle internalization study using fluorescent polystyrene beads, where it was reported that a low uptake (∼2%) was achieved for 250 nm beads at an incubation concentration of 80,000 nanoparticles/cell.35 The negative surface charge (ζ-potential) of PLGA nanoparticle used in our study could in part be implicated in the observed low intracellular uptake. However, it has been shown previously that the nonspecific adsorption of serum proteins from culture medium can modify the surface charge of nanoparticles and induce their receptor mediated endocytosis.36 We hypothesize that a similar uptake mechanism is likely relevant to our PLGA nanoparticles. Our microRNA loading, release, and cell internalization results show that the nontargeted PLGA nanoparticles used in our study can still accumulate in sufficient quantities inside glioblastoma cells for subsequent efficient downregulation. This information has significant merit in its own right because it highlights the desirability for additional chemical modifications to actively target PLGA nanoparticles to the cells of interest, to thus increase even more the ratio of nanoparticles internalized by the cells without the need to use high concentrations of nanoparticles in the incubation medium. Functionalizing the surface of nanoparticles to surface targets of cells of interest would also be more relevant in the clinical setting. Indeed, this is the subject of our ongoing research in this area, requiring the detailed analysis of multiple additional new parameters relevant to cell targeting and anti-microRNA therapy. Future studies will also investigate all the above factors, and the generalizability of the proposed concept of tailored anti-microRNA therapy, using other antisense microRNAs, other cell lines, and, importantly, human glioblastoma derived cells. Owing to the significantly higher copy number of miR-21 molecules (>25,000) compared to miR-10b (