Micelles In Cells Research Articles

Construction of esterase-responsive hyperbranched polyprodrug micelles and their antitumor activity in vitro

Abstract This research constructs an esterase-responsive hyperbranched polyprodrug nano pharmaceutical and investigates their antitumor activity. Polyprodrug micelle was prepared by one-pot method based on glutathione (GSH), doxorubicin (DOX), and polyethylene glycol (PEG) under the catalyst of N,N-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and 1-hydroxybenzotriazole (HOBt). The polyprodrug was characterized by nuclear magnetic resonance (NMR), Fourier transform infrared spectrometer (FT-IR), ultraviolet-visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), and transmission electron microscope (TEM), respectively. The antitumor activity of polyprodrug micelle was evaluated by Hela cell and the distributions of micelles in cells were observed by fluorescent microscope. The NMR and FT-IR confirmed that the DOX-GSH-PEG polyprodrug was successfully synthesized. The drug loading rate is 10.21% and particle size is 106.4 ± 1 nm with a narrowed polydispersity (PDI = 0.145). The DLS showed that the micelles were stable during 7 days at 25°C. The drug release results showed that the micelles could be esterase-responsive disrupted, and the drug release rate could reach 43% during 72 h. Cell uptake and cell viability demonstrated that the micelles could distribute to cell nuclei during 8 h and induce cell apoptosis during 48 h. Overall, these hyperbranched polyprodrug micelles prepared by one-pot method could be esterase-responsive disrupted and release the antitumor drugs in a high esterase environment for cancer therapy in vitro. These results confirm that DOX-GSH-PEG is an effective nanomedicine in vitro and the endogenous-based strategy with one-pot synthesis to construct esterase-responsive polyprodrug would probably be a preferred choice in the future.

Regulating the uptake of poly(N-(2-hydroxypropyl) methacrylamide)-based micelles in cells cultured on micropatterned surfaces.

Cellular uptake of nanoparticles plays a crucial role in cell-targeted biomedical applications. Despite abundant studies trying to understand the interaction between nanoparticles and cells, the influence of cell geometry traits such as cell spreading area and cell shape on the uptake of nanoparticles remains unclear. In this study, poly(vinyl alcohol) is micropatterned on polystyrene cell culture plates using ultraviolet photolithography to control the spreading area and shape of individual cells. The effects of these factors on the cellular uptake of poly(N-(2-hydroxypropyl)methacrylamide)-based micelles were investigated at a single-cell level. Human carcinoma MCF-7 and A549 cells as well as normal Hs-27 and MRC-5 fibroblasts were cultured on micropatterned surfaces. MCF-7 and A549 cells, both with larger sizes, had a higher total micelle uptake. However, the uptake of Hs-27 and MRC-5 cells decreased with increasing spreading area. In terms of cell shapes, MCF-7 and A549 cells with round shapes showed a higher micelle uptake, while those with a square shape had a lower cellular uptake. On the other hand, Hs-27 and MRC-5 cells showed opposite behaviors. The results indicate that the geometry of cells can influence the nanoparticle uptake and may shed light on the design of functional nanoparticles.

Detection of Lipase Activity in Cells by a Fluorescent Probe Based on Formation of Self-Assembled Micelles.

SummaryReliable and sensitive detection of lipase activity is essential for the early diagnosis and monitoring of acute pancreatitis or progression of digestive diseases. However, the available fluorescent probes for detection of lipase activity are only implemented in a hexane-water two-phase system due to the nature of heterogeneous catalysis of lipase, thus limiting their applications in direct imaging of lipase activity in living cells and tissues. Here we designed and synthesized a “turn on” fluorescent probe CPP based on self-assembled micelles for hydrolysis of lipase. The CPP probe exhibits high selectivity and excellent sensitivity for the detection of lipase in such a homogeneous system and is successfully applied for monitoring lipase activity in pancreatic AR42J cells, tissues, and serums. Taken together, the fluorescent CPP probe not only provides a tool for diagnostic potential in pancreatic disease but also demonstrates an application potential for micelle self-assembly-based development of biological probes.

Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study.

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7).

Docetaxel-Loaded Self-Assembly Stearic Acid-Modified Bletilla striata Polysaccharide Micelles and Their Anticancer Effect: Preparation, Characterization, Cellular Uptake and In Vitro Evaluation.

Poorly soluble drugs have low bioavailability after oral administration, thereby hindering effective drug delivery. A novel drug-delivery system of docetaxel (DTX)-based stearic acid (SA)-modified Bletilla striata polysaccharides (BSPs) copolymers was successfully developed. Particle size, zeta potential, encapsulation efficiency (EE), and loading capacity (LC) were determined. The DTX release percentage in vitro was determined using high performance liquid chromatography (HPLC). The hemolysis and in vitro anticancer activity were studied. Cellular uptake and apoptotic rate were measured using flow cytometry assay. Particle size, zeta potential, EE and LC were 125.30 ± 1.89 nm, −26.92 ± 0.18 mV, 86.6% ± 0.17%, and 14.8% ± 0.13%, respectively. The anticancer activities of DTX-SA-BSPs copolymer micelles against HepG2, HeLa, SW480, and MCF-7 (83.7% ± 1.0%, 54.5% ± 4.2%, 48.5% ± 4.2%, and 59.8% ± 1.4%, respectively) were superior to that of docetaxel injection (39.2% ± 1.1%, 44.5% ± 5.3%, 38.5% ± 5.4%, and 49.8% ± 2.9%, respectively) at 0.5 μg/mL drug concentration. The DTX release percentage of DTX-SA-BSPs copolymer micelles and docetaxel injection were 66.93% ± 1.79% and 97.06% ± 1.56% in two days, respectively. Cellular uptake of DTX-FITC-SA-BSPs copolymer micelles in cells had a time-dependent relation. Apoptotic rate of DTX-SA-BSPs copolymer micelles and docetaxel injection were 73.48% and 69.64%, respectively. The SA-BSPs copolymer showed good hemocompatibility. Therefore, SA-BSPs copolymer can be used as a carrier for delivering hydrophobic drugs.

RGD peptide-mediated chitosan-based polymeric micelles targeting delivery for integrin-overexpressing tumor cells

BackgroundSolid tumors need new blood vessels to feed and nourish them as well as to allow tumor cells to escape into the circulation and lodge in other organs, which is termed “angiogenesis.” Some tumor cells within solid tumors can overexpress integrins αvβ3 and αvβ5, which can specifically recognize the peptide motif Arg-Gly-Asp (RGD). Thus, the targeting of RGD-modified micelles to tumor vasculature is a promising strategy for tumor-targeting treatment.MethodsRGD peptide (GSSSGRGDSPA) was coupled to poly(ethylene glycol)-modified stearic acid-grafted chitosan (PEG-CS-SA) micelles via chemical reaction in the presence of N,N′-Disuccinimidyl carbonate. The critical micelle concentration of the polymeric micelles was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. The micelle size, size distribution, and zeta potential were measured by light scattering and electrophoretic mobility. Doxorubicin (DOX) was chosen as a model anticancer drug to investigate the drug entrapment efficiency, in vitro drug-release profile, and in vitro antitumor activities of drug-loaded RGD-PEG-CS-SA micelles in cells that overexpress integrins (ανβ3 and ανβ5) and integrin-deficient cells.ResultsUsing DOX as a model drug, the drug encapsulation efficiency could reach 90%, and the in vitro drug-release profiles suggested that the micelles could be used as a controlled-release carrier for the hydrophobic drug. Qualitative and quantitative analysis of cellular uptake indicated that RGD-modified micelles could significantly increase the DOX concentration in integrin-overexpressing human hepatocellular carcinoma cell line (BEL-7402), but not in human epithelial carcinoma cell line (Hela). The competitive cellular-uptake test showed that the cellular uptake of RGD-modified micelles in BEL-7402 cells was significantly inhibited in the presence of excess free RGD peptides. In vitro cytotoxicity tests demonstrated DOX-loaded RGD-modified micelles could specifically enhance the cytotoxicity against BEL-7402 compared with DOX-loaded PEG-CS-SA and doxorubicin hydrochlorate.ConclusionThis study suggests that RGD-modified PEG-CS-SA micelles are promising drug carriers for integrin-overexpressing tumor active targeting therapy.

Electrostatic effects on the migration rates of ions between charged micelles. Consequences for fluorescence quenching

The rate of exchange of countenons between micelles has been calculated from numerical solutions of the Poisson-Boltzmann equation for micelles in cells of spherical symmetry, and solutions of a steady-state diffusion problem for the ion in the resulting potential. The results indicate a rapid increase of the exchange rate with total amphiphilc concentration, as has been observed.