Evaluating Nanomedicines: Obstacles and Advancements.

Continued advancements in nanotechnology are expanding the boundaries of medical research, most notably as drug delivery agents for treatment against cancer. Drug delivery with nanotechnology can offer greater control over the biodistribution of therapeutic agents to improve the therapeutic index. In the last 20 years, a number of nanomedicines have transitioned into the clinic. As nanomedicines evolve, techniques to properly evaluate their safety and efficacy must also evolve. Characterization methods for nano-based materials must be adapted to the demands of nanomedicine developers and regulators. This second edition book provides updated characterization protocols designed to address the clinical potential of nanomedicines during their preclinical development. In this chapter, the characterization challenges of nanoparticles intended for drug delivery will be discussed, along with examples of advancements and improvements in nanomedicine characterization.

Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells.

UNLABELLED: Liver fibrosis is a common outcome of chronic liver disease that leads to liver cirrhosis and hepatocellular carcinoma. No US Food and Drug Administration-approved targeted antifibrotic therapy exists. Activated hepatic stellate cells (aHSCs) are the major cell types responsible for liver fibrosis; therefore, eradication of aHSCs, while preserving quiescent HSCs and other normal cells, is a logical strategy to stop and/or reverse liver fibrogenesis/fibrosis. However, there are no effective approaches to specifically deplete aHSCs during fibrosis without systemic toxicity. aHSCs are associated with elevated expression of death receptors and become sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. Treatment with recombinant TRAIL could be a potential strategy to ameliorate liver fibrosis; however, the therapeutic application of recombinant TRAIL is halted due to its very short half-life. To overcome this problem, we previously generated PEGylated TRAIL (TRAILPEG ) that has a much longer half-life in rodents than native-type TRAIL. In this study, we demonstrate that intravenous TRAILPEG has a markedly extended half-life over native-type TRAIL in nonhuman primates and has no toxicity in primary human hepatocytes. Intravenous injection of TRAILPEG directly induces apoptosis of aHSCs in vivo and ameliorates carbon tetrachloride-induced fibrosis/cirrhosis in rats by simultaneously down-regulating multiple key fibrotic markers that are associated with aHSCs. CONCLUSION: TRAIL-based therapies could serve as new therapeutics for liver fibrosis/cirrhosis and possibly other fibrotic diseases. (Hepatology 2016;64:209-223).

What is the future of PEGylated therapies?

The tremendous potential of biologic drugs is hampered by short half-lives in vivo, resulting in significantly lower potency than activity seen in vitro. These short-acting therapeutic agents require frequent dosing profiles that can reduce applicability to the clinic, particularly for chronic conditions. Therefore, half-life extension technologies are entering the clinic to enable improved or new biologic therapies. PEGylation is the first successful technology to improve pharmacokinetic (PK) profiles of therapeutic agents and has been applied in the clinic for over 25 years. Over 10 PEGylated therapeutics have entered the clinic since the early 1990 s, and new PEGylated agents continue to expand clinical pipelines and drug patent life. PEGylation is the most established half-life extension technology in the clinic with proven safety in humans for over two decades. Still, it is one of the most evolving and emerging technologies that will be applied for the next two decades.

Delivery of tumor-homing TRAIL sensitizer with long-acting TRAIL as a therapy for TRAIL-resistant tumors.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has attracted great interest as a cancer therapy because it selectively induces death receptor (DR)-mediated apoptosis in cancer cells while sparing normal tissue. However, recombinant human TRAIL demonstrates limited therapeutic efficacy in clinical trials, possibly due to TRAIL-resistance of primary cancers and its inherent short half-life. Here we introduce drug delivery approaches to maximize in vivo potency of TRAIL in TRAIL-resistant tumor xenografts by (1) extending the half-life of the ligand with PEGylated TRAIL (TRAILPEG) and (2) concentrating a TRAIL sensitizer, selected from in vitro screening, in tumors via tumor-homing nanoparticles. Antitumor efficacy of TRAILPEG with tumor-homing sensitizer was evaluated in HCT116 and HT-29 colon xenografts. Western blot, real-time PCR, immunohistochemistry and cell viability assays were employed to investigate mechanisms of action and antitumor efficacy of the combination. We discovered that doxorubicin (DOX) sensitizes TRAIL-resistant HT-29 colon cancer cells to TRAIL by upregulating mRNA expression of DR5 by 60% in vitro. Intravenously administered free DOX does not effectively upregulate DR5 in tumor tissues nor demonstrate synergy with TRAILPEG in HT-29 xenografts, but rather introduces significant systemic toxicity. Alternatively, when DOX was encapsulated in hyaluronic acid-based nanoparticles (HAC/DOX) and intravenously administered with TRAILPEG, DR-mediated apoptosis was potentiated in HT-29 tumors by upregulating DR5 protein expression by 70% and initiating both extrinsic and intrinsic apoptotic pathways with reduced systemic toxicity compared to HAC/DOX or free DOX combined with TRAILPEG (80% vs. 40% survival rate; 75% vs. 34% tumor growth inhibition). This study demonstrates a unique approach to overcome TRAIL-based therapy drawbacks using sequential administration of a tumor-homing TRAIL sensitizer and long-acting TRAILPEG.

Self-assembly of gold nanoparticles to silver microspheres as highly efficient 3D SERS substrates.

Herein we report a simple, one-pot, surfactant-free synthesis of 3D Ag microspheres (AgMSs) in aqueous phase at room temperature. The 3D AgMSs act as supports to fix the gold nanoparticles (GNPs) in 3D space via the interaction between the carboxyl groups of GNPs and the Ag atoms of AgMSs. The ensemble of AgMSs@GNPs with high surface-enhanced Raman scattering (SERS) activity and sensitivity can be an ideal 3D substrate choice for practical SERS detection applications. The simple self-assembly strategy may be extended to other metallic materials with great potentials in SERS, catalysis, and photoelectronic devices.

Mesenchymal stem cell-based cell engineering with multifunctional mesoporous silica nanoparticles for tumor delivery.

Stem cell engineering, the manipulation and control of cells, harnesses tremendous potential for diagnosis and therapy of disease; however, it is still challenging to impart multifunctionalization onto stem cells to achieve both. Here we describe a mesenchymal stem cell (MSC)-based multifunctional platform to target orthotopic glioblastoma by integrating the tumor targeted delivery of mesenchymal stem cells and the multimodal imaging advantage of mesoporous silica nanoparticles (MSNs). Rapid cellular uptake, long retention time and stability of particles exemplify the potential that the combination of MSNs and MSCs has as a stem cell-based multifunctional platform. Using such a platform, we verified tumor-targeted delivery of MSCs by in vivo multimodal imaging in an orthotopic U87MG glioblastoma model, displaying higher tumor uptake than particles without MSCs. As a proof-of-concept, this MSC platform opens a new vision for multifunctional applications of cell products by combining the superiority of stem cells and nanoparticles for actively targeted delivery.

Highly robust, recyclable displacement assay for mercuric ions in aqueous solutions and living cells.

We designed a recyclable Hg(2+) probe based on Rhodamine B isothiocyanate (RBITC)-poly(ethylene glycol) (PEG)-comodified gold nanoparticles (AuNPs) with excellent robustness, selectivity, and sensitivity. On the basis of a rational design, only Hg(2+) can displace RBITC from the AuNP surfaces, resulting in a remarkable enhancement of RBITC fluorescence initially quenched by AuNPs. To maintain stability and monodispersity of AuNPs in real samples, thiol-terminated PEG was employed to bind with the remaining active sites of AuNPs. Besides, this displacement assay can be regenerated by resupplying free RBITC into the AuNPs solutions that were already used for detecting Hg(2+). Importantly, the detection limit of this assay for Hg(2+) (2.3 nM) was lower than the maximum limits guided by the United States Environmental Protection Agency as well as that permitted by the World Health Organization. The efficiency of this probe was demonstrated in monitoring Hg(2+) in complex samples such as river water and living cells.

A facile, one-step nanocarbon functionalization for biomedical applications.

Despite their immense potential in biomedicine, carbon nanomaterials suffer from inefficient dispersion and biological activity in vivo. Here we utilize a single, yet multifunctional, hyaluronic acid-based biosurfactant to simultaneously disperse nanocarbons and target single-walled carbon nanotubes (SWCNTs) to CD44 receptor positive tumor cells with prompt uptake. Cellular uptake was monitored by intracellular enzyme-activated fluorescence, and localization of SWCNTs within cells was further confirmed by Raman mapping. In vivo photoacoustic, fluorescence, and positron emission tomography imaging of coated SWCNTs display high tumor targeting capability while providing long-term, fluorescence molecular imaging of targeted enzyme events. By utilizing a single biomaterial surfactant for SWCNT dispersion without additional bioconjugation, we designed a facile technique that brings nanocarbons closer to their biomedical potential.

Noninvasive monitoring of orthotopic glioblastoma therapy response using RGD-conjugated iron oxide nanoparticles.

Noninvasive imaging techniques have been considered important strategies in the clinic to monitor tumor early response to therapy. In the present study, we applied RGD peptides conjugated to iron oxide nanoparticles (IONP-RGD) as contrast agents in magnetic resonance imaging (MRI) to noninvasively monitor the response of a vascular disrupting agent VEGF(121)/rGel in an orthotopic glioblastoma model. RGD peptides were firstly coupled to IONPs coated with a crosslinked PEGylated amphiphilic triblock copolymer. In vitro binding assays confirmed that cellular uptake of particles was mainly dependent on the interaction between RGD and integrin α(v)ß(3) of human umbilical vein endothelial cells (HUVEC). The tumor targeting of IONP-RGD was observed in an orthotopic U87 glioblastoma model. Finally, noninvasive monitoring of the tumor response to VEGF(121)/rGel therapy at early stages of treatment was successfully accomplished using IONP-RGD as a contrast agent for MRI, a superior method over common anatomical approaches which are based on tumor size measurements. This preclinical study can accelerate anticancer drug development and promote clinical translation of nanoprobes.

Long-term multimodal imaging of tumor draining sentinel lymph nodes using mesoporous silica-based nanoprobes.

The imaging of sentinel lymph nodes (SLNs), the first defense against primary tumor metastasis, has been considered as an important strategy for noninvasive tracking tumor metastasis in clinics. In this study, we report the development and application of mesoporous silica-based triple-modal nanoprobes that integrate multiple functional moieties to facilitate near-infrared optical, magnetic resonance (MR) and positron emission tomography (PET) imaging. After embedding near-infrared dye ZW800, the nanoprobe was labeled with T(1) contrast agent Gd(3+) and radionuclide (64)Cu through chelating reactions. High stability and long intracellular retention time of the nanoprobes was confirmed by in vitro characterization, which facilitate long-term in vivo imaging. Longitudinal multimodal imaging was subsequently achieved to visualize tumor draining SLNs up to 3 weeks in a 4T1 tumor metastatic model. Obvious differences in uptake rate, amount of particles, and contrast between metastatic and contra-lateral sentinel lymph nodes were observed. These findings provide very helpful guidance for the design of robust multifunctional nanomaterials in SLNs' mapping and tumor metastasis diagnosis.

Protease-activated drug development.

In this extensive review, we elucidate the importance of proteases and their role in drug development in various diseases with an emphasis on cancer. First, key proteases are introduced along with their function in disease progression. Next, we link these proteases as targets for the development of prodrugs and provide clinical examples of protease-activatable prodrugs. Finally, we provide significant design considerations needed for the development of the next generation protease-targeted and protease-activatable prodrugs.

Sticky nanoparticles: a platform for siRNA delivery by a bis(zinc(II) dipicolylamine)-functionalized, self-assembled nanoconjugate.

Delivering the goods: Multifunctional, self-assembled, polymeric nanoparticles for the simultaneous delivery of small-molecule drugs and siRNA have been synthesized. The nanoparticles are composed of biodegradable hyaluronic acid, for tumor targeting and cellular delivery, and a high siRNA binding affinity is provided by a Zn(II)-dipicolylamine analogue as an artificial phosphate-binding receptor (see scheme).

High-sensitivity nanosensors for biomarker detection.

High sensitivity nanosensors utilize optical, mechanical, electrical, and magnetic relaxation properties to push detection limits of biomarkers below previously possible concentrations. The unique properties of nanomaterials and nanotechnology are exploited to design biomarker diagnostics. High-sensitivity recognition is achieved by signal and target amplification along with thorough pre-processing of samples. In this tutorial review, we introduce the type of detection signals read by nanosensors to detect extremely small concentrations of biomarkers and provide distinctive examples of high-sensitivity sensors. The use of such high-sensitivity nanosensors can offer earlier detection of disease than currently available to patients and create significant improvements in clinical outcomes.

Dual-functional, receptor-targeted fluorogenic probe for in vivo imaging of extracellular protease expressions.

Herein, we report a new type of in vivo fluorogenic probe that enables simultaneous and active targeting of overexpressed receptors, α(V)ß(3) integrins, and extracellular proteases, matrix metalloproteinases (MMPs), in the tumor regions. This c(RGDyK)-conjugated MMP fluorogenic probe efficiently targets the tumor regions with high retention time while maintaining receptor binding affinity and substrate activity. The probe minimizes nonspecific accumulation, thus demonstrating improved tumor-to-background signal ratio (T/N) in both α(V)ß(3) integrin- and MMP-overexpressing U87MG tumor-bearing mouse model. This strategy can be easily tuned for a wide array of applications targeting various receptors and extracellular proteases in vivo.

Real-time video imaging of protease expression in vivo.

We demonstrate the first true real-time in vivo video imaging of extracellular protease expression using an ultrafast-acting and extended-use activatable probe. This simple, one-step technique is capable of boosting fluorescent signals upon target protease cleavage as early as 30 minutes from injection in a small animal model and is able to sustain the strong fluorescent signal up to 24 hours. Using this method, we video imaged the expression and inhibition of matrix metalloproteinases (MMPs) in a tumor-bearing mouse model. The current platform can be universally applied to any target protease of interest with a known peptide substrate and is adaptable to a wide range of real-time imaging applications with high throughputs such as for in vivo drug screening, examinations of the therapeutic efficacy of drugs, and monitoring of disease onset and development in animal models.

The design and application of fluorophore-gold nanoparticle activatable probes.

Fluorescence-based assays and detection techniques are among the most highly sensitive and popular biological tests for researchers. To match the needs of research and the clinic, detection limits and specificities need to improve, however. One mechanism is to decrease non-specific background signals, which is most efficiently done by increasing fluorescence quenching abilities. Reports in the literature of theoretical and experimental work have shown that metallic gold surfaces and nanoparticles are ultra-efficient fluorescence quenchers. Based on these findings, subsequent reports have described gold nanoparticle fluorescence-based activatable probes that were designed to increase fluorescence intensity based on a range of stimuli. In this way, these probes can detect and signify assorted biomarkers and changes in environmental conditions. In this review, we explore the various factors and theoretical models that affect gold nanoparticle fluorescence quenching, explore current uses of activatable probes, and propose an engineering approach for future development of fluorescence based gold nanoparticle activatable probes.

Inorganic nanoparticles for multimodal molecular imaging.

Multimodal molecular imaging can offer a synergistic improvement of diagnostic ability over a single imaging modality. Recent development of hybrid imaging systems has profoundly impacted the pool of available multimodal imaging probes. In particular, much interest has been focused on biocompatible, inorganic nanoparticle-based multimodal probes. Inorganic nanoparticles offer exceptional advantages to the field of multimodal imaging owing to their unique characteristics, such as nanometer dimensions, tunable imaging properties, and multifunctionality. Nanoparticles mainly based on iron oxide, quantum dots, gold, and silica have been applied to various imaging modalities to characterize and image specific biologic processes on a molecular level. A combination of nanoparticles and other materials such as biomolecules, polymers, and radiometals continue to increase functionality for in vivo multimodal imaging and therapeutic agents. In this review, we discuss the unique concepts, characteristics, and applications of the various multimodal imaging probes based on inorganic nanoparticles.

Molecular imaging of cell-based cancer immunotherapy.

Cell-based cancer immunotherapy represents a new and powerful weapon in the arsenal of anticancer treatments. Non-invasive monitoring of the disposition, migration and destination of therapeutic cells will facilitate the development of cell based therapy. The therapeutic cells can be modified intrinsically by a reporter gene or labeled extrinsically by introducing imaging probes into the cells or on the cell surface before transplant. Various advanced non-invasive molecular imaging techniques are playing important roles in optimizing cellular therapy by tracking cells and monitoring the therapeutic effects of transplanted cells in vivo. This review will summarize the application of multiple molecular imaging modalities in cell-based cancer immunotherapy.