Tf-conjugated Nanoparticles Research Articles

Transferrin-Decorated PLGA Nanoparticles Loaded with an Organoselenium Compound as an Innovative Approach to Sensitize MDR Tumor Cells: An In Vitro Study Using 2D and 3D Cell Models.

Multidrug resistance (MDR) is the main challenge in cancer treatment. In this sense, we designed transferrin (Tf)-conjugated PLGA nanoparticles (NPs) containing an organoselenium compound as an alternative to enhance the efficacy of cancer therapy and sensitize MDR tumor cells. Cytotoxicity studies were performed on different sensitive tumor cell lines and on an MDR tumor cell line, and the Tf-conjugated NPs presented significantly higher antiproliferative activity than the nontargeted counterparts in all tested cell lines. Due to the promising antitumor activity of the Tf-decorated NPs, further studies were performed using the MDR cells (NCI/ADR-RES cell line) comparatively to one sensitive cell line (HeLa). The cytotoxicity of NPs was evaluated in 3D tumor spheroids and, similarly to the results achieved in the 2D assays, the Tf-conjugated NPs were more effective at reducing the spheroid's growth. The targeted Tf-NPs were also able to inhibit tumor cell migration, presented a higher cell internalization and induced a greater number of apoptotic events in both cell lines. Therefore, these findings evidenced the advantages of Tf-decorated NPs over the nontargeted counterparts, with the Tf-conjugated NPs containing an organoselenium compound representing a promising drug delivery system to overcome MDR and enhance the efficacy of cancer therapy.

Therapeutic Targeting of the RAS-Pathway by Synthetic Mir-181a Nanoparticles in Acute Myeloid Leukemia (AML).

Abstract 2422Today most AML patients (pts) still fail to achieve long-term survival. New therapeutic options are needed. We and others reported that high miR-181a expression associated with better outcomes in AML pts (Marcucci et al NEJM 2008; Schwind et al JCO 2010; Li et al Blood 2012), but the underlying mechanisms remain unclear. Aberrant RAS activation by mutations or overexpression is frequent in human malignancies, including AML. Previously KRAS was described as a direct miR-181a target in oral squamous cell carcinoma (Shin et al BBRC 2011). Here we confirmed KRAS as a direct miR-181a target & identified miR-181a-binding sites in the MAPK1 untranslated region (UTR) utilizing in silico tools & consequently identified MAPK1 as a new direct miR-181a target of the RAS signaling pathway. We observed respectively a 30±5% (±standard deviation; P=.003) & 35±2% (P=.0002) downregulation of luciferase activity after co-transfecting 293T cells with miR-181a mimic vs scramble control (sc) molecules and luciferase reporter constructs spanning the predicted miR-181a binding sites (seed sequences) in KRAS or MAPK1. Mutations in the seed sequences of KRAS or MAPK1 could rescue the miR-181a mimic induced downregulation. In the KG1a & OCI-AML3 AML cells with low endogenous miR-181a level but activated RAS-signaling, lentiviral miR-181a overexpression reduced KRAS protein by 2.8 & 2.1 fold and MAPK1 by 2.8 & 1.5 fold (normalized to β-actin & quantified by densitometry) respectively compared to sc expressing controls. Consistent with this result, lentiviral-based knock-down of miR-181a increased KRAS by 1.4 & 3.2 fold and MAPK1 protein by 2.3 & 1.8 fold in KG1a & OCI-AML3 cells respectively, compared to sc controls.Since higher miR-181a associated with improved outcomes &miR-181a targets the RAS-pathway in AML, increasing miR-181a may have therapeutic value for AML pts. We formulated transferrin (Tf) targeted anionic lipid based lipopolyplex nanoparticles (NP) to deliver synthetic miR-181a mimic molecules to AML cells which overexpress Tf receptors, while overcoming RNA degradation & facilitating the cellular uptake of the miR molecules. Mature miR-181a in KG1a, OCI-AML3 cells & pts blasts (n=3) were measured by qPCR after treatment with Tf conjugated NP (TfNP) encapsulating miR-181a mimic molecules (TfNP181a) or sc (TfNPsc) at 10 nM. After 24h mature miR-181a levels increased 211±31, 880±10 & 145±19 fold in KG1a, OCI-AML3 & pts blasts (n=3), while levels of miR-181b & unrelated miR-140 remained unchanged. We found that delivered miR-181a downregulated KRAS & MAPK1 proteins (KG1a: 2.5 & 2.1 fold; OCI-AML3: 2.5 & 2.4 fold; pts blasts: 9.0 & 5.8 fold respectively) compared to TfNPsc treatment. To analyze downstream effects of TfNP181a mediated inhibition of the RAS pathway we assessed the expression of total MEK & AKT and p-MEK & p-AKT. TfNP181a compared to TfNPsc treatment decreased p-MEK by 3.2, 2.1 & 5 fold and p-AKT by 2.9, 3.6 & 6.5 fold in KG1a, OCI-AML3 & pts blasts. Furthermore, a 3.2, 4.7 & 3.1 fold reduction of the transcription factor MYC, which is regulated by MAPK in KG1a, OCI-AML3 & pts blasts treated with TfNP181a vs TfNPsc was observed.TfNP181a treatment also resulted in antileukemic activity. TfNP181a compared to TfNPsc treatment led to reduced proliferation of KG1a (34±8% at day 4, P=.01) & OCI-AML3 (31±4% at day 4, P=.02) cells, and decreased colony formation (after 2 weeks) in both cell lines by 40±5% (P<.01) & 44±5% (P<.0001) respectively. Since higher miR-181a levels associated with improved treatment response we next tested whether TfNP181a could sensitize AML cells to daunorubicin (DNR). After 24h priming with 10 nM TfNP181a or TfNPsc, KG1a & OCI-AML3 cells were treated with 0.2 μM DNR for 72h. MTS assays showed decreased viability of the cells primed with TfNP181a vs TfNPsc: 66% vs 80.6% (P=.01) and 58% vs 81% (P=.02) for KG1a & OCI-AML3 respectively. We confirmed these findings in pts blasts primed with TfNP181a: 62.4% vs TfNPsc: 96.5% (P<.01). Preclinical in vivo tests of TfNP181a are ongoing. In conclusion we first report that miR-181a targets KRAS and its downstream effector MAPK1 in AML. TfNP181a treatment resulted in reduced RAS expression & signaling inhibition, antileukemic activity & increased sensitivity to chemotherapy in AML cells. Developing TfNP181a for miR-181a replacement therapy may represent a novel therapeutic approach to AML & may be extended to other malignancies involving RAS signaling. Disclosures:No relevant conflicts of interest to declare.

Targeted Delivery of MicroRNA-29b by Nanoparticles Provides Antileukemic Activity and Increases Sensitivity to the Hypomethylating Agent Decitabine (DAC) in Acute Myeloid Leukemia (AML)

Abstract 81Despite advances in our understanding of disease mechanisms the outcome of the majority of AML patients (pts) remains poor. Epigenetic gene silencing by DNA hypermethylation has been shown to contribute to AML. Recently we demonstrated promising results of treatment with the hypomethylating agent DAC in older AML pts. Pts with higher miR-29b expression were more likely to respond to DAC. MiR-29b targets DNA methyltransferases (DNMTs; i.e. DNMT1, DNMT3A & DNMT3B) and has hypomethylating and tumor suppressor activity in AML. Thus uncovering a method to increase intracellular miR-29b and sensitize AML blasts to DAC may be of therapeutic value. Free synthetic miRs are degraded in bio-fluid and have very limited cellular uptake. To overcome these limitations, we developed a novel non-viral delivery system for synthetic miRs. Polyethylenimine was used to capture synthetic miRs & form a polyplex core. An outside layer of lipid components, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine, Linoleic acid & 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (MW=2,000) was used to protect the miRs from degradation & clearance and facilitate their uptake. Using this approach we produced nanoparticles (NPs) encapsulating 0.4 μM synthetic miR-29b precursors (pM29b) or scramble controls (sc) [both Ambion]. Since AML blasts and cell lines overexpress the transferrin receptor, we conjugated the NPs with transferrin (Tf) to increase the uptake by targeted delivery. To evaluate the efficiency of NP uptake we encapsulated FAM-fluorescent labeled-pM29b and treated AML cell lines (i.e. Kasumi-1 & MV4-11). Tf-conjugated NP (TfNP) treated cells showed the highest uptake by confocal microscopy and flow-cytometry (mean fluorescence intensity (MFI): 21.0 & 22.9 for Kasumi-1 & MV4-11 respectively) compared to non-Tf-conjugated NPs (NP; MFI: 8.3 & 13.0). To evaluate if pM29b was processed into its mature form we measured levels of mature miR-29b in MV4-11 & Kasumi-1 by qPCR after treatment with TfNP encapsulating pM29b (TfNP29b) or NP encapsulating pM29b (NP29b). At 48h mature miR-29b increased 2000 and 2100 fold after treatment with TfNP29b but only 20 and 50 fold following treatment with NP29b in MV4-11 & Kasumi-1 respectively, compared to untreated or TfNPsc treated cells. Thus TfNPs were 40–100 times more efficient than NPs in increasing levels of miR-29b. Our observations were validated in primary blasts from 3 AML pts. At 48h miR-29b levels increase on average 400 fold in TfNP29b vs TfNPsc treated blasts. The levels of an unrelated miR (i.e. miR-140) remained unchanged in all our experiments. These results demonstrate that pM29b is efficiently delivered and processed into its mature form after TfNP delivery, leading to a specific increase of miR-29b in AML cell lines & primary blasts with otherwise low endogenous miR-29b expression. Downregulation of the miR-29b targets DNMT1, DNMT3A, and DNMT3B on RNA & protein level, determined by qPCR and western blotting respectively, was observed in both cell lines & primary AML samples at 48 hours following TfNP29b compared to TfNPsc treatment. The observed fold reduction on RNA level for DNMT1 was 0.63, 0.53 & 0.63, for DNMT3A 0.48, 0.53 & 0.58, and for DNMT3B 0.60, 0.73 & 0.53 in treated MV4-11, Kasumi-1 cells & pts blasts (mean, n=3), respectively. Increased miR-29b, following TfNP29b treatment resulted in antileukemic activity. Treatment with TfNP29b compared to TfNPsc led to 30% & 35% reduction in proliferation at day 5 and 50% & 64% decreased colony forming ability (scored after two weeks) in MV4-11 & Kasumi-1 cells, respectively. To investigate a possible sensitizing effect of miR-29b to DAC treatment, we treated MV4-11 cells and primary AML blasts with 2.5 uM DAC, which is comparable to concentrations achievable in treated AML pts. 72 hours after DAC treatment, decreased viability (measured by MTS assays) was observed in cells treated with TfNP29b (for 48h) followed by DAC compared with those treated with TfNPsc followed by DAC: 36% vs 53% (p<0.001) for MV4-11 cells and 63% vs 73% (p<0.01) for primary pts blasts. In conclusion, we report an effective nanoparticle-based delivery system for miRs. Our delivery system for pM29b showed in vitro antileukemic activity and improved response to DAC in AML cells, demonstrating that miR-29b is not only a predictor for clinical response to DAC, but may also functions as a novel therapeutic, sensitizer to hypomethylating agents in AML. Disclosures:No relevant conflicts of interest to declare.

Efficacy of transferrin-conjugated paclitaxel-loaded nanoparticles in a murine model of prostate cancer.

Chemotherapy remains the preferred choice of treatment for prostate cancer but modest drug response and significant toxicity by conventional methods of administration limit their efficacy. In our study, we determined the efficacy of paclitaxel (Tx)-loaded biodegradable nanoparticles (NPs) on tumor inhibition. We hypothesized that NPs following conjugation to transferrin (Tf) ligand (NPs-Tf) would enhance the therapeutic efficacy of the encapsulated drug. The antiproliferative activity of NPs was determined in human prostate cancer cell line (PC3) and their effect on tumor inhibition in a murine model of prostate cancer. NPs (approximately 220 nm in diameter, 5.4% w/w drug loading) under in vitro conditions exhibited sustained release of the encapsulated drug (60% release in 60 days). The IC50 (concentration of drug for 50% inhibition of cell growth) of the drug with Tf-conjugated NPs (Tx-NPs-Tf) was about 5-fold lower than that with unconjugated NPs (Tx-NPs) or drug in solution. Animals that received a single-dose intratumoral injection of Tx-NPs-Tf (Tx dose= 4 mg/kg) demonstrated complete tumor regression and greater survival rate than those that received either Tx-NPs or Tx-Cremophor EL formulation. In conclusion, sustained release NPs demonstrated greater antitumor activity following their conjugation to Tf ligand.