Recent Advances in pH-Sensitive Polymeric Nanoparticles for Smart Drug Delivery in Cancer Therapy.

BACKGROUND: Rapid developments in the field of nanoparticles during the last decades have led to its increased application in drug delivery approaches. The advantages of nano-based drug delivery systems include improved therapeutic efficacy of drugs and the reduction of side effects. OBJECTIVE: This review aims to highlight advances in stimuli-triggered drug delivery approaches using polymeric nanoparticles with a focus on pH-sensitive drug- and theranostic delivery systems. RESULTS AND CONCLUSION: Of the various organic/inorganic nanoparticles, polymeric nanoparticles have fulfilled an integral role in the advancement of drug delivery systems by virtue of the ease to incorporate and modify targeting moieties in combination with controlled drug release over prolonged periods. Furthermore, polymeric nanoparticles facilitate theranostic treatment by the incorporation of imaging agents in addition to therapeutics. Recently, stimuli-responsive polymeric nanoparticles emerged as smart drug carriers, in which drug release is affected by physical and/or chemical structural changes induced by a specific stimulus (e.g. pH, temperature, and specific enzymes). The use of these nanocomposites reduces premature drug release and maintains effective drug levels at the pathological target.

Antioxidant activity of levan coated cerium oxide nanoparticles.

Levan coated cerium oxide nanoparticles (LCNPs) with the enhanced antioxidant activity were successfully synthesized and characterized. Levan and their derivatives are attractive for biomedical applications attributable to their antioxidant, anti-inflammation and anti-tumor properties. LCNPs were synthesized using the one-pot and green synthesis system with levan. For production of nanoparticles, levan plays a role as a stabilizing and reducing agent. Fourier transform infrared spectroscopy (FT-IR) analysis showed that LCNPs successfully synthesized. The morphology and size of nanoparticles were confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). LCNPs have good water solubility and stability. The conjugation of levan with cerium oxide nanoparticles improved antioxidant activity. Moreover the level of ROS was reduced after treatment of LCNPs to H2O2 stimulated NIH3T3 cells. These results demonstrate that the LCNPs are useful for applying of treatment of ROS induced diseases.

Preparation of pyrenyl-based multifunctional nanocomposites for biomedical applications.

Nanocomposites are widely used to obtain an accurate diagnosis of, and to provide effective therapy for, a number of diseases, because they can be easily formulated by introducing therapeutic agents (e.g., drugs and genes) and imaging agents (e.g., magnetic nanocrystals). Furthermore, nanocomposites can be developed as all-in-one systems, which enable cancer diagnosis and therapy, as well as the simultaneous monitoring of drug behavior. In this protocol, we describe the synthesis of four pyrenyl-based polymers (pyrenyl polyethylene glycol (Py-PEG), pyrenyl dextran (Py-DEX), pyrenyl hyaluronan (Py-HA) and pyrenyl-conjugated heterofunctional PEG (pyrenyl PEG)) and their subsequent use in the preparation of multifunctional nanocomposites for different applications including multimodal imaging, targeted cancer detection and pH-sensitive drug delivery. Notably, these nanocomposites can be used to simultaneously perform multiple tasks--for example, delivering magnetic particles for early cancer detection by MRI, efficient cataloging of patient groups for personalized therapy and real-time monitoring of disease progress. Starting from the synthesis of pyrenyl-based polymers, this protocol can be completed in ∼15 d.

Simple, rapid detection of influenza A (H1N1) viruses using a highly sensitive peptide-based molecular beacon.

A peptide-based molecular beacon (PEP-MB) was prepared for the simple, rapid, and specific detection of H1N1 viruses using a fluorescence resonance energy transfer (FRET) system. The PEP-MB exhibited minimal fluorescence in its "closed" hairpin structure. However, in the presence of H1N1 viruses, the specific recognition of the hemagglutinin (HA) protein of H1 strains by the PEP-MB causes the beacon to assume an "open" structure that emits strong fluorescence. The PEP-MB could detect H1N1 viruses within 15 min or even 5 min and can exhibit strong fluorescence even at low viral concentrations, with a detection limit of 4 copies.

A scanometric antibody probe for facile and sensitive immunoassays.

We have developed a novel scanometric antibody probe for rapid, sensitive, and naked-eye-visible immunoassays. Using this probe, we clearly demonstrated the successful scanometric detection and identification of influenza A viruses on a microarray. In addition, the sensitivity of the scanometric immunoassay was comparable to that of the fluorescence-based method.

Photo-crosslinkable hydrogel-based 3D microfluidic culture device.

We developed the photo-crosslinkable hydrogel-based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo-crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 µm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular-shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel-based 3D microfluidic device, showing that 53-75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo-crosslinkable hydrogel-based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.

Self-assembled levan nanoparticles for targeted breast cancer imaging.

We report on the targeted imaging of breast cancer using self-assembled levan nanoparticles. Indocyanine green (ICG) was encapsulated in levan nanoparticles via self-assembly. Levan-ICG nanoparticles were found to be successfully accumulated in breast cancer via specific interaction between fructose moieties in levan and overexpressed glucose transporter 5 in breast cancer cells.

Live imaging of cellular dynamics using a multi-imaging vector in single cells.

Real-time monitoring of cellular dynamics in living organisms is highly challenging. We developed a multi-imaging vector based on 2A peptides. Live imaging of subcellular compartments can be performed following the transfection of cells with another vector, the multi-labeling vector, which contains localization signals and various fluorescent protein variants.

In vivo imaging of islet transplantation using PLGA nanoparticles containing iron oxide and indocyanine green.

PURPOSE: We determined whether poly(lactic-co-glycolic acid) nanoparticles would be a useful reagent for the successful monitoring of isolated islets by magnetic resonance imaging and optical imaging systems, without clinically relevant toxicity in vitro or in vivo. METHODS: We used iron oxide for MR imaging and a cyanide dye approved by the Food and Drug Administration (indocyanine green) for optical imaging and estimated the in vivo detection of transplanted pancreatic islets. RESULTS: The poly(lactic-co-glycolic acid) nanoparticles were associated with the islets in vitro and were successfully detected by 4.7 T (MR) and optical imaging, without other toxic effects. When labeled islets were transplanted under the mouse kidney capsule, in vivo T2/ T2*-weighted scans with 4.7 T MR detected as few as 300 labeled islets by 4 weeks. Optical in vivo imaging revealed indocyanine green fluorescence by 2 and 4 days after transplantation of islets containing 250 and 500 µg/mL poly(lactic-co-glycolic acid) nanoparticles, respectively. These results were further supported by the immunohistochemical results for insulin and iron in the recipient mouse kidney and pancreas. CONCLUSIONS: Taken together, these data indicate that poly(lactic-co-glycolic acid) nanoparticles may be used to label transplanted islets and may be imaged with in vivo MR and optical imaging systems.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment.

Multiplexed detection of various breast cancer cells by perfluorocarbon/quantum dot nanoemulsions conjugated with antibodies.

The effective targeting of cancer cell surface antigens is an attractive approach in cancer diagnosis and therapy. Multifunctional nanoprobes with cell-targeting specificity are likely to find important applications in bioanalysis, biomedicine, and clinical diagnosis. In this study, we have fabricated biocompatible perfluorocan/quantum dot nanoemulsions as bimodal imaging nanoprobes for the targeting of breast cancer cells. Perfluorocarbon/quantum dot nanoemulsions conjugated with monoclonal antibodies, as a type of bimodal imaging nanoprobe based on 19 F-MR and optical imaging, have been synthesized and applied for targeted imaging of three different breast cancer cells (SKBR3, MCF-7, MDA-MB 468), respectively. We have shown that the cancer-detection capabilities of antibody-conjugated PFC/QDs nanoemulsions could be successfully applied to target of various breast cancer cells. These modified PFC/QDs nanoemulsions were shown to target the cancer cell surface receptors specially. Conjugation of ligands to nanoemulsions targeting over-expressed cell surface receptors is a promising approach for targeted imaging to tumor cells. We further propose that the PFC/QDs nanoemulsions could be used in targeted imaging of breast cancer cells.

Anchoring foreign substances on live cell surfaces using Sortase A specific binding peptide.

A simple, rapid, and efficient live cell surface labeling method has been developed that uses a direct conjugation between Sortase A expressed transiently at the cell surface and Sortase A specific binding peptide.

A simple, fast and highly sensitive assay for the detection of telomerase activity.

A highly sensitive and rapid PCR-free telomerase activity assay has been developed that uses SYBR Green intercalation into the G-quadruplex structures in the presence of K(+).

Bimodal perfluorocarbon nanoemulsions for nasopharyngeal carcinoma targeting.

PURPOSE: The aim of this study was to perform the detection of folate receptor (FR)-positive tumors with a bimodal imaging contrast agent, a perfluorocarbon (PFC)/rhodamine nanoemulsion, providing both 19F-based magnetic resonance imaging (MRI) and fluorescence imaging capabilities. PROCEDURES: The PFC/rhodamine nanoemulsion was further infused with phospholipid-anchored folate to improve the ability to target FR-expressing tumors. The preferential accumulation of the FR-targeted bimodal nanoemulsion in FR-positive tumor sites was monitored by both 19F-MRI and optical imaging. RESULTS: The FR-targeted PFC nanoemulsion had no significant effect on cell viability, and the size and fluorescence signal of PFC nanoemulsion were very stable. These nanoprobes were successfully delivered into FR-positive tumor xenograft models and showed significantly enhanced signal intensities of 19F-MRI and fluorescence imaging in the tumor area. CONCLUSIONS: The folate-PFC/rhodamine nanoemulsion has a great potential to serve as a useful optical and 19F-MRI agent for the diagnosis and targeting of FR-positive tumor.

Novel cell penetrating peptides with multiple motifs composed of RGD and its analogs.

Cell penetrating peptides (CPPs) have been used to transport macromolecules into cells. Most CPPs have properties such as a strong polycationic charge, amphipathic basic, and hydrophobicity. In this study, we designed the peptides with multiple motifs composed of RGD and its analogs to induce integrin-mediated endocytosis as well as endosomal escape by forming an amphipathic helix in acidic endosomes. These peptides were proved less toxic to animal cells than those without acidic residues. Unexpectedly, peptide conjugated liposomes could penetrate into cells regardless of integrins. The replacement of all aspartic acids by glutamic acids did not prevent the peptide-mediated liposome uptake, and the higher basic and leucine contents enhanced the gene silencing activity of siRNA encapsulated in the liposomes. The peptide is considered to be a new type of CPP which can be used for drug delivery.

Bifunctional nanoparticles constructed using one-pot encapsulation of a fluorescent polymer and magnetic (Fe3O4) nanoparticles in a silica shell.

Integration of biocompatible silica with a fluorescent polymer (PDDF) and superparamagnetic iron oxide nanoparticles (Fe3 O4 ) to form uniform core-shell nanostructures has the great potential to form particles for use in multimodal bioimaging applications. Core-shell nanoparticles (PDDF/Fe3 O4 @SiO2 ) exhibit fluorescent and magnetic properties that are favorable for their use in magnetic separation and guiding applications, as well as optical and magnetic resonance (MR) imaging capabilities. With the biological analysis in an in vitro intracellular permeation and cytotoxicity test, chemical conjugation of the surface using folic acid (FA) molecules can provide the nanoparticles with cell-targeting properties, localizing the nanoparticles to folate receptors (FRs) on target KB cells that over-express the FRs.

Folate-conjugated cross-linked magnetic nanoparticles as potential magnetic resonance probes for in vivo cancer imaging.

Superparamagnetic iron oxide nanoparticles are widely used as nanoprobes for magnetic resonance imaging (MRI). In this study, a novel type of cross-linked magnetic nanoparticle was developed in an effort to improve the structural stability of amphiphilic polymer-coated iron oxide nanoparticles. Iron oxide nanocrystals were coated with a cross-linkable amphiphilic graft copolymer, poly(succinimide) grafted with folate-conjugated polyethylene glycol (PEG) and alkyl chains. The tumor-specific targeting ligand, folate, was included to target and detect cancer cells. The hydrophobic portions of the amphiphilic copolymer on the surfaces of the nanoparticles were cross-linked via an aminolysis reaction between the succinimide units and a bifunctional primary amine. The folate-conjugated cross-linked magnetic nanoparticles (F-CLMNPs) were 40 nm in diameter and displayed a low cytotoxicity, even at relatively high concentrations. The F-CLMNPs exhibited highly efficient intracellular uptake into KB cells, which overexpress the folate receptor, as determined by fluorescence microscopy, flow cytometry, and Prussian blue staining. In vivo MR images of a mouse bearing a KB cell tumor displayed a 75% drop in the T2 signal in the tumor tissues within 3 hours. These results indicated that the F-CLMNPs accumulated at the tumor site and were highly effective for tumor detection using in vivo MRI techniques.

Differential in vitro and cellular effects of iron chelators for hypoxia inducible factor hydroxylases.

Hypoxia inducible factor 1α (HIF-1α), an essential transcriptional factor, is negatively regulated by two different types of oxygen and Fe(2+) -dependent HIF hydroxylases, proline hydroxylase (PHD) and factor inhibiting HIF (FIH), under normoxia. Iron chelators have therefore been used for inducing HIF-1α expression by inhibiting the hydroxylases. In this study, the iron chelators displayed differential effects for PHD and FIH in cells depending on their iron specificity and membrane permeability rather than their in vitro potencies. The membrane permeability of the strict Fe(2+) -chelator potentially inhibited both hydroxylases, whereas the membrane impermeable one showed no inhibitory effect in cells. In contrast, the depletion of the extracellular Fe(3+) ion was mainly correlated to PHD inhibition, and the membrane permeable one elicited low efficacy for both enzymes in cells. The 3'-hydroxyl group of quercetin, a natural flavonoid, was critical for inhibition of intracellular hydroxylases. Since the 3'-methylation of quercetin is induced by catechol-O-methyl transferase, the enzyme may regulate the intracellular activity of quercetin. These data suggest that the multiple factors of iron-chelators may be responsible for regulating the intracellular activity HIF hydroxylases.

Cascade imaging of proteolytic pathways in cancer cells using fluorescent protein-conjugated gold nanoquenchers.

The real-time monitoring of the caspase cascade in cancer cells during apoptosis was performed using various caspase substrate-linked fluorescent proteins-conjugated gold nanoparticles as a new imaging probe.

Indocyanine green encapsulated nanogels for hyaluronidase activatable and selective near infrared imaging of tumors and lymph nodes.

Indocyanine green (ICG) encapsulated hyaluronic acid (HA) nanogels were first studied for highly selective detection of specific cancers and lymph nodes via hyaluronidase sensitive switch-on of near infrared fluorescence as a long-lasting and stimuli-responsive imaging probe.