Ethlinn V.B. van Gaal

Ligand-targeted Particulate Nanomedicines Undergoing Clinical Evaluation: Current Status

Since the introduction of Doxil® on the market nearly 20years ago, a number of nanomedicines have become part of treatment regimens in the clinic. With the exception of antibody-drug conjugates, these nanomedicines are all devoid of targeting ligands and rely solely on their physicochemical properties and the (patho)physiological processes in the body for their biodistribution and targeting capability. At the same time, many preclinical studies have reported on nanomedicines exposing targeting ligands, or ligand-targeted nanomedicines, yet none of these have been approved at this moment. In the present review, we provide a concise overview of 13 ligand-targeted particulate nanomedicines (ligand-targeted PNMs) that have progressed into clinical trials. The progress of each ligand-targeted PNM is discussed based on available (pre)clinical data. Main conclusions of these analyses are that (a) ligand-targeted PNMs have proven to be safe and efficacious in preclinical models; (b) the vast majority of ligand-targeted PNMs is generated for the treatment of cancer; (c) contribution of targeting ligands to the PNM efficacy is not unambiguously proven; and (d) targeting ligands do not cause localization of the PNM within the target tissue, but rather provide benefits in terms of target cell internalization and target tissue retention once the PNM has arrived at the target site. Increased understanding of the in vivo fate and interactions of the ligand-targeted PNMs with proteins and cells in the human body is mandatory to rationally advance the clinical translation of ligand-targeted PNMs. Future perspectives for ligand-targeted PNM approaches include the delivery of drugs that are unable or inefficient in passing cellular membranes, treatment of drug resistant tumors, targeting of the tumor blood supply, the generation of targeted vaccines and nanomedicines that are able to cross the blood-brain barrier.

Constrained and UV-activatable cell-penetrating peptides for intracellular delivery of liposomes.

Herein we report on the development of a novel method of constraining a cell-penetrating peptide, which can be used to trigger transport of liposomes into cells upon in this case radiation with UV-light. A cell-penetrating peptide, which was modified on both termini with an alkyl chain, was anchored to the liposomal surface in a constrained and deactivated form. Since one of the two alkyl chains was connected to the peptide via a UV-cleavable linker, disconnection of this alkyl chain upon irradiation led to the exposure of the cell-penetrating peptide, and mediated the transport of the entire liposome particle into cells.

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles

Abstract Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications. From the Clinical Editor The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology.

How to screen non-viral gene delivery systems in vitro?

Screening of new gene delivery candidates regarding transfection efficiency and toxicity is usually performed by reading out transgene expression levels relative to a reference formulation after in vitro transfection. However, over the years and among different laboratories, this screening has been performed in a variety of cell lines, using a variety of conditions and read-out systems, and by comparison to a variety of reference formulations. This makes a direct comparison of results difficult, if not impossible. Reaching a consensus would enable placing new results into context of previous findings and estimate the overall contribution to the improvement of non-viral gene delivery. In this paper we illustrate the sensitivity of transfection outcomes on testing conditions chosen, and propose a screening protocol with the aim of standardization within the field.

Plasmid engineering for controlled and sustained gene expression for nonviral gene therapy

Gene therapy requires the introduction of genetic material in diseased cells with the aim of treating or ultimately curing a disease. Since the start of gene therapy clinical trials in 1990, gene therapy has proven to be possible, but studies to date have highlighted the difficulty of achieving efficient, specific, and long-term transgene expression. Efforts to improve gene therapy strategies over the past years were mainly aimed at solving the problem of delivery, without paying much attention to the optimization of the expression cassette. With the current understanding of the eukaryotic transcription machinery and advanced molecular biology techniques at our disposition, it has now become possible to create custom-made transgene expression cassettes optimized for gene therapy applications. In this review, we will discuss several strategies that have been explored to improve the level and duration of transgene expression, to increase control over expression, or to restrict transgene expression to specific cell types or tissues. Although still in its infancy, such strategies will eventually lead to improvement of nonviral gene therapy and expansion of the range of possible therapeutic applications.

Decationized crosslinked polyplexes for redox-triggered gene delivery.

The clinical applicability of polymers as gene delivery systems depends not only on their efficiency, but also on their safety. The cytotoxicity of these systems remains a major issue, mainly due to their cationic nature. Therefore, it is highly preferable to have a system based on biocompatible neutral polymers, lacking polycations, without compromising the DNA condensing and protecting capacities. Here, we introduce a concept to obtain a neutral polymeric gene delivery system, through a 3-step process (charge-driven condensation; stabilization through disulfide crosslinking; polyplex decationization) to generate polyplexes with a core of disulfide crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) in which plasmid DNA (pDNA) is entrapped and a shell of poly(ethylene glycol) (PEG). The resulting polyplexes combine beneficial features of high and stable DNA loading capacity, stealth behavior and reduced toxicity. The nanoparticles are designed to release the pDNA after cellular uptake through cleavage of disulfide crosslinks within the intracellular reducing environment. This was shown by forced introduction of the polyplexes into the cytosol of HeLa cells by electroporation, which resulted in a high level of expression of the reporter gene. Additionally, the decationized polyplexes showed no interference on the cellular cell viability or metabolic activity (even at high dose) and no complex-induced membrane destabilization. Furthermore, decationized polyplexes showed a low degree of non-specific uptake, which is a highly favorable property for targeted therapy. Summarizing, the stabilized, decationized polyplexes presented here contribute to solve the high toxicity, low stability and lack of cellular/tissue specificity of cationic polymer based gene delivery systems.

DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

ABSTRACTPurposeTo evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.MethodsA set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.ResultsNeither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.ConclusionNo beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.

Junk DNA enhances pEI-based non-viral gene delivery

Gene therapy aims at delivering exogenous DNA into the nuclei of target cells to establish expression of a therapeutic protein. Non-viral gene delivery is examined as a safer alternative to viral approaches, but is presently characterized by a low efficiency. In the past years several non-viral delivery strategies have been developed, including cationic polymer-based delivery. One of the most described and most active polymers is linear pEI. This study addresses questions regarding formulating highly efficient pEI-based polyplexes. By mixing reporter plasmid DNA with non-coding junk DNA it was shown that the amount of reporter plasmid can be significantly decreased in linear pEI-based transfection while maintaining transfer activity. Junk DNA maximally exerts its function when co-delivered with active DNA within the same pEI complexes rather than upon co-delivery of distinct junk DNA/pEI and active DNA/pEI complexes. We conclude that not the total amount of active DNA, but rather the total amount of active DNA-containing particles is the limiting factor in pEI-mediated transfection.

A novel approach for the intravenous delivery of leuprolide using core-cross-linked polymeric micelles

Therapeutic peptides are highly attractive drugs for the treatment of various diseases. However, their poor pharmacokinetics due to rapid renal elimination limits their clinical applications. In this study, a model hormone peptide, leuprolide, was covalently linked to core-cross-linked polymeric micelles (CCL-PMs) via two different hydrolysable ester linkages, thereby yielding a nanoparticulate system with tuneable drug release kinetics. The ester linkage that provided the slowest peptide release kinetics was selected for in vivo evaluation. Compared to the soluble peptide, the leuprolide-entrapped CCL-PMs showed a prolonged circulation half-life (14.4h) following a single intravenous injection in healthy rats and the released leuprolide was detected in blood for 3days. In addition, the area under the plasma concentration-time curve (AUC) value was >100-fold higher for leuprolide-entrapped CCL-PMs than for soluble leuprolide. Importantly, the released peptide remained biologically active as demonstrated by increased and long-lasting plasma testosterone levels. This study shows that covalent linkage of peptides to CCL-PMs via hydrolytically sensitive ester bonds is a promising approach to achieving sustained systemic levels of peptides after intravenous administration.

Tailoring the physicochemical properties of core-crosslinked polymeric micelles for pharmaceutical applications

To optimally exploit the potential of (tumor-) targeted nanomedicines, platform technologies are needed in which physicochemical and pharmaceutical properties can be tailored according to specific medical needs and applications. We here systematically customized the properties of core-crosslinked polymeric micelles (CCPM). The micelles were based on mPEG-b-pHPMAmLacn (i.e. methoxy poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate]), similar to the block copolymer composition employed in CriPec® docetaxel, which is currently in phase I clinical trials. The CCPM platform was tailored with regard to size (30 to 100nm), nanocarrier degradation (1month to 1year) and drug release kinetics (10 to 90% in 1week). This was achieved by modulating the molecular weight of the block copolymer, the type and density of the crosslinking agent, and the hydrolytic sensitivity of the drug linkage, respectively. The high flexibility of CCPM facilitates the development of nanomedicinal products for specific therapeutic applications.