Determination Of Cellular Uptake Research Articles

Abstract B87: Mechanistic insights into the antitumor activity of SB 11285—a novel STING agonist

Abstract Background: Cancer immunotherapy is a highly effective therapeutic option for cancer patients. However, the overall response rate with checkpoint inhibitors and other related modalities has been modest. Targeting innate immune signaling pathways that induce type I IFN production to reprogram tumor microenvironment and restore antitumor immunity represents a novel immunotherapeutic approach. We have previously reported that SB 11285 is a first-in-class synthetic cyclic dinucleotide STING agonist, which has demonstrated potent antitumor activity, when used alone or combined with other antitumor agents, in several syngeneic mouse and rat tumor models when administered by intratumoral, intravenous or intraperitoneal routes. Presented here are studies that provide additional insights into the mechanism of action of SB 11285 and analogs. Methods: (a) SB 11285 induces Type I IFN production and other cytokines in human PBMCs and PBMC-derived monocytes. PBMCs and monocytes, isolated from fresh PBMCs using pan monocyte isolation kit (Miltenyi Biotec), were stimulated with SB 11285. Type I IFNs and other cytokines were quantified using regular and multiplex ELISA assays. (b) SB 11285 directly binds STING. To address whether SB 11285 directly binds wild-type human STING, surface plasmon resonance assay was performed with a Biacore T200 device and a biotinylated SB 11285 analog (Biot-SB 11285). (c) Activity against STING polymorphs. Evaluation of SB 11285 and analogs was carried out using both SZ14 reporter cells (HEK293-derived) and HEK293T cells that stably or transiently express STING polymorphic variants. (d) Pharmacodynamic studies. Normal BALB/c mice were injected intravenously with SB 11285 or its analogs at 9 mg/kg. Serum, spleen, and liver samples were collected to quantify RANTES and TNF-α using ELISA. The expression of representative ISGs in spleen samples, including IRF7, IFIT2, and OAS1b were quantified using real-time PCR. (e) Determination of cellular uptake of SB 11285 by PBMCs. The STING agonist activity of Biot-SB 11285 in inducing type I IFN response was confirmed using SZ14 reporter cells and THP1-Dual-WT reporter cells. Human PBMCs were incubated with Biot-SB 11285 and the cellular uptake of compound by immune cells was evaluated using flow cytometry. Results and Conclusion: Our studies provide significant mechanistic insights into the STING agonistic activity of SB 11285 and analogs in that they: (a) induce Type I IFNs, other cytokines and chemokines in PBMCs and monocytes, monocyte-derived dendritic cells; (b) directly bind STING with nanomolar affinity; (c) activate multiple human STING polymorphic variants; (d) induce cytokines, chemokines, and ISGs in mice following i.v. injection; and (e) are effectively taken up by monocytes and other immune cells. SB 11285 is being advanced to human clinical trials. Citation Format: Shenghua Zhou, Sreerupa Challa, Vishal Nair, Geeta Meher, Anjaneyulu Sheri, Rayomand Gimi, Seetharamaiyer Padmanabhan, Dillon Cleary, Leena Suppiah, Diane Schmidt, Santosh Khedkar, Radhakrishnan Iyer. Mechanistic insights into the antitumor activity of SB 11285—a novel STING agonist [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B87.

Determination of Cellular Uptake and Endocytic Pathways

Efficiency of drug and gene delivery via nonviral vehicles is contingent on proper cellular uptake and intracellular release. Further, various cargos, such as nucleases for gene editing or inhibitors for endosomal receptors, require transport to specific compartments of the cell. Hence, characterization of cellular uptake and endocytic pathways is crucial for the optimization of any nanoparticle-mediated intracellular delivery system. Previous work on endocytic pathways looks at the effect of various pathway inhibitors on the uptake efficiency of nanoparticles carrying fluorescently-labeled cargo. While this helps attribute particle uptake to specific pathways like caveolae-mediated or clathrin-mediated endocytosis, this does not provide a holistic picture of the delivery process. Here, we provide a general protocol that combines systematic studies of inhibitor effects on efficiency with quantification of particle-induced cell membrane permeability. By applying this methodology to a nucleic acid delivery system, for example a helical polypeptide-based nanoparticle for plasmid and guide RNA delivery, we gain understanding of the endocytic mechanisms and cell uptake for intelligent design of intracellular delivery.

Nickel Oxide Nanoparticles Induce Oxidative DNA Damage and Apoptosis in Kidney Cell Line (NRK-52E).

Increasing use of nickel oxide (NiO) nanoparticles in different applications results in high occupational and environmental exposure to them. However, the effect of NiO nanoparticles on human health is still poorly documented. It was aimed to investigate the toxic potentials of NiO nanoparticles on NRK-52E kidney epithelial cells. The following assays were used: the nanoparticle characterization by transmission electron microscopy (TEM) and dynamic light scattering (DLS); the determination of cellular uptake and morphologic changes by TEM and inductively coupled plasma-mass spectrometry (ICP-MS); MTT and neutral red uptake (NRU) assays for cytotoxicity; comet assay for genotoxicity; the determination of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyl (PC) and glutathione (GSH) levels by enzyme-linked immune sorbent assays (ELISA) for the potential of oxidative damage; and Annexin V-FITC apoptosis detection assay with propidium iodide (PI) for apoptosis. The nanoparticles were taken up by the cells and induced dose-dependent DNA damage by comet assay and oxidative damage evidenced by increasing levels of MDA, 8-OHdG, PC and depletion of GSH. At ≥294.0μg/mL concentration, NiO nanoparticles caused 50% inhibition in cell viability by the cytotoxicity assays. Also, they showed apoptotic/necrotic effects on the cells as well as some morphological changes. We have indicated that their cellular damage effects should raise concern about the safety associated with their applications in consumer products.

Modeling uptake of nanoparticles in multiple human cells using structure–activity relationships and intercellular uptake correlations

Biomedical applications of nanoparticles (NPs) are largely dependent on their cellular uptake potential that enables them to reach the specific targets in the body. Experimental determination of cellular uptake of diverse functionalized NPs in different human cell types is tedious, expensive and time intensive, hence compelling for alternative methods. We developed quantitative structure–activity relationship (QSAR) models for predicting uptake of functionalized NPs in multiple cell types in accordance with the OECD guidelines. The decision treeboost QSAR models precisely predicted uptake of 104 NPs in five different cell types yielding high R2 between experimental and model predicted values in the respective training (>0.966) and test (>0.914) sets. The cross-validation Q2 values ranged between 0.627 and 0.926. Low RMSE (<0.11) and MAE (<0.09) in test data emphasized for the usefulness of developed models for predicting new NPs, which outperformed the previous reports. Relevant structural features of NPs (modifier) that were responsible and influence the cellular permeability were identified. Here, we also attempted to develop intercellular uptake correlations based quantitative activity–activity relationship (QAAR) models for predicting cellular viability of NPs for all the cell types. The performances of all the 20 developed QAAR models were highly comparable with the QSAR models. The applicability domains of the developed models were defined using leverage method. The proposed QAAR models can be employed for extrapolating activity endpoints of NPs to either of the five cell types when the data for the other cell type are available. The developed models can be used as tools for screening new functionalized NPs for their cell-specific affinities prior to their biomedical applications.

Copper (II) Oxide Nanoparticles Induced Nephrotoxicity In Vitro Conditions

Copper (II) oxide nanoparticles (CuO-NPs) have been used in industry, cosmetics, and medicine. Gradually increased human exposure to CuO-NPs can cause undesirable effects in humans and the ecosystem. Although a few studies indicate that CuO-NPs can damage organs/systems, we still lack data on the toxicological effects of CuO-NPs in kidneys. In the present study, the nephrotoxic potential of CuO-NPs was investigated in in vitro conditions. The following assays were used: transmission electron microscopy and inductively coupled plasma mass spectrometry for particle characterization and determination of cellular uptake; MTT and neutral red uptake assays for cytotoxicity; comet assay for genotoxicity; enzyme-linked immune sorbent assay (ELISA) determination of malondialdehyde (MDA), 8-hydroxy-deoxyguanosine (8-OHdG), protein carbonyl (PC), and glutathione (GSH) levels for oxidative damage; and Annexin V-FITC apoptosis detection assay with propidium iodide (PI) for apoptosis. Our results show that CuO-NPs (34.9 nm) caused significant cytotoxicity (IC50: 11.6–16.4 μg/mL), genotoxicity (1.9- to 8.4-fold), and oxidative and apoptotic effects in the range of 2.5–80 μg/mL in kidney cells. In conclusion, CuO-NPs are hypothesized to negatively affect human health and caused nephrotoxicity. However, further studies should be better to elucidate their toxic mechanism.

In Vitro Toxicological Assessment of Magnesium Oxide Nanoparticle Exposure in Several Mammalian Cell Types.

Worldwide researchers have rising concerns about magnesium-based materials, especially magnesium oxide (MgO) nanaoparticles, due to increasing usage as promising structural materials in various fields including cancer treatment. However, there is a serious lack of information about their toxicity at the cellular and molecular levels. In this study, the toxic potentials of MgO nanoparticles were investigated on liver (HepG2), kidney (NRK-52E), intestine (Caco-2), and lung (A549) cell lines. For the toxicological assessment, the following assays were used: the particle characterization by transmission electron microscopy, the determination of cellular uptake by inductively coupled plasma-mass spectrometry, MTT and neutral red uptake assays for cytotoxicity, comet assay for genotoxicity, and the determination of malondialdehyde (MDA), 8-hydroxydeoxyguanosine, protein carbonyl, and glutathione levels by enzyme-linked immune sorbent assays for the potential of oxidative damage and annexin V-fluorescein isothiocyanate (FITC) apoptosis detection assay with propidium iodide (PI) for apoptosis. Magnesium oxide nanoparticles were taken up by the cells depending on their concentration and agglomeration/aggregation potentials. Magnesium oxide nanoparticles induced DNA (≤14.27 fold) and oxidative damage. At a concentration of ≥323.39 µg/mL, MgO nanoparticles caused 50% inhibition in cell viability by 2 different cytotoxicity assays. The cell sensitivity to cytotoxic and genotoxic damage induced by MgO nanoparticles was ranked as HepG2 < A549 < Caco-2 < NRK-52E. Although it was observed that MgO nanoparticles induced apoptotic effects on the cells, apoptosis was not the main cell death. DNA damage, cell death, and oxidative damage effects of MgO nanoparticles should raise concern about the safety associated with their applications in consumer products.

The new InsP3Kinase inhibitor BIP-4 is competitive to InsP3 and blocks proliferation and adhesion of lung cancer cells

As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20μM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157±57nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3μM or 2μM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells.

Determination of cellular uptake and intracellular levels of Cenersen (Aezea®, EL625), a p53 antisense oligonucleotide in acute myeloid leukemia cells

TP53 encodes for tumor protein p53. The suppression of p53 protein results in interruption of DNA repair mechanisms in dividing malignant cells thereby increasing the DNA damage and activating p53-independent mechanisms of apoptosis. This ultimately may translate into enhanced cytotoxic effects of standard chemotherapy. Based on this rationale, Cenersen, a phosphorothioate oligonucleotide antisense to p53-mRNA was synthesized and tested in clinical trials for patients with acute myeloid leukemia (AML). An important component of Cenersen clinical development is to develop a sensitive and specific method to quantify plasma and intracellular levels of Cenersen in different biologic matrices in order to determine tissue and intracellular distribution of the parent compound and its metabolites. Ultimately, this will allow us to determine pharmacokinetic and pharmacodynamic relationship for dose–effect correlation and design effective regimen to be rapidly translate into the clinic. An ELISA-based assay was adapted for assay development and validation of Cenersen in mouse plasma and cell lysate. Cellular uptake of Cenersen was studied in MV4-11 and KASUMI-1 AML cell lines. Real-time RT-PCR was used to measure P53-mRNA expression changes in treated cells. The assay had a limit of quantification of 35pmol/L in mouse plasma. Within-day and between-day precision of <15% and accuracy nearly 100% were observed in a linear range of 10–2000pmol/L (R2=0.99) in AML cell lysate. The selectivity of this assay examined as cross-reactivity with its 3′N-1, 3′N-2-metabolites, was 16.8% and 0.4%, respectively, and with its mismatch and the scramble oligonucleotides was 0.06% and 0.4%, respectively. Cenersen was stable in mouse plasma up to 8h at 37°C. When exposed to 0.1–1μmol/L Cenersen, MV4-11 and KASUMI-1 cells showed intracellular concentration in the range of 9.97–45.34nmol/mg protein and 0.1–2.1nmol/mg protein, respectively. Successful downregulation of p53-mRNA expression was observed in Cenersen treated cells. This ELISA-based assay was applicable to plasma and intracellular concentration measurement of Cenersen. Assessment of achievable concentration of Cenersen in different biologic matrices will be useful to elucidate the biological and clinical activity of this promising drug and define its recommended dose in future clinical trials.

The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [14C]putrescine or [14C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (Kt = 24.58 +/- 2.24 microM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 +/- 0.43 microM, Kt' = 0.61 +/- 0.10 microM). From the Ki values, it is inferred that N1-5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N1-acetylspermine, and then N1,N12-diacetylspermine. N1-acetylspermidine and N8-acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows: N1,N12-diacetylspermine approximately N1-5-amino-2-nitro-benzoylspermine > N1-acetylspermine. Both N1-acetylspermidine (Ki = 753 +/- 25 microM, Ki' = 128 +/- 5 microM) and N8-acetylspermidine (Ki = 22.4 +/- 0.4 microM, Ki' = 279 +/- 3 microM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N4,N9-diacetylspermine, nor N1,N4-bis(beta-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.

Adriamycin cellular transport: Methodological aspects

A rapid and simple method for determination of cellular uptake of adriamycin is described. The method is based on the principle that active uptake is proportional to alterations of drug distribution, measured as a fraction of time, between suspending medium and cells, the volume of each having been accurately determined. Cellular drug uptake can be calculated by the use of a simple distribution formula. This method represents a compromise between indirect measurement of the loss of drug from suspending medium and direct measurement of drug uptake following cell separation, washing, and lysis. This method should be applicable to the measurement of cellular uptake of a wide range of drugs.