Escaping the endosome: evaluating the mechanisms of cellular trafficking of non-viral vehicles (2023)

Fluorescent nanosensor-based tumor imaging holds great promise in cancer diagnosis and treatment support, however, signal contrast is severely impaired by unspecific/unwanted activation in microscopic regions with a severely limited local abundance of biomarkers. Here we developed an activation enhancement strategy by integrating and manipulating the coactivation of two lysosomal detection and escape factors, facilitating increased accessibility of the cytosolic biomarker. Using hybrid DNA probes in gold nanoquenchers, ATP detection triggered a conformational switch of the corresponding aptamer units, triggering the exposure of a hidden loop in a loop structure. Subsequently, miRNA-21 recognition was triggered by toehold-mediated strand displacement and shedding of binding complexes. Application of the lysosomotropic agent chloroquine at optimized time intervals facilitated the release of nanosensors into the cytosol and an approximately 10.5-fold increase in intracellular fluorescencein vitro, while coactivation improved the signaling rate from cancer cells to normal cells by 5.9 times. The synergistic effects resulted in a high tumor-to-normal tissue ratio of approximately 7.9 imliveimage results. This strategy establishes a new paradigm of fluorescent nanosensors for selective and specific tumor imaging.

In the field of nucleic acid delivery, there has long been skepticism as to whether or not polycationic drug delivery systems would make it into clinical practice. However, with the disclosure of patents on polyethyleneimine-based RNA transporters by leaders in the field of nucleic acid therapeutics such as BioNTech SE, and with clinical trials moving beyond Phase I trials, this reluctance appears to be diminishing. As one of the most distinguishing properties of polymer-based arrays, exceptional adjustability can be both a blessing and a curse. Knowing the setscrews and how they affect drug carrier performance gives the formulation scientist committed to their development a head start. Here we provide the reader with a toolbox - a toolbox intended to advise and assist the developer in designing a state-of-the-art poly or micellar system for the delivery of therapeutic nucleic acids; specifically, to manipulate the vector for maximum endosomal escape performance with minimal toxicity. So, after briefly outlining the constraints of polymer vector design, let's dive into the topic of endosomal delivery. Not only will we discuss the most recent findings about the endolysosomal compartment, but we will also present several hypotheses and mechanisms that facilitate endosomal escape from polyplex systems. Finally, we will combine the different facets presented in the previous chapters with the basic building blocks of polymer vector design and evaluate the advantages and disadvantages. Throughout the article, special emphasis is placed on cellular specificities, not only as an additional barrier, but also as inspiration for how these cell-specific properties can be exploited.

Over the past two decades, nanoparticle delivery systems have revolutionized cancer treatment, achieving targeted delivery, enhanced bioavailability, and an improved toxicity profile. The growing interest in nanotechnology for the treatment of cancer stems from the unique physicochemical properties of nanoparticles (e.g. small size, surface properties,etc.). Indeed, several anti-cancer drugs can now be effectively delivered through nano-delivery systems. However, the application of such delivery systems in the field of gene therapy is still in its infancy. Furthermore, the treatment of retinoblastoma (RB), an aggressive childhood eye cancer, is a major concern in developing countries due to the late diagnosis of this type of cancer. While delivery strategies based on adeno-associated viruses remain the mainstay of the gene delivery method due to their high efficiency, other delivery systems such as e.g. g., non-viral nanoparticles (NPs), have been developed as alternative therapeutic modalities. Indeed, various nanoparticle formulations such as lipid-based nanoparticles, polymer nanoparticles, and gold nanoparticles have shown improved gene delivery efficiency in retinal diseases. This review article focuses on nanoparticle-mediated gene therapy approaches in the treatment of RB and highlights the attempts made to develop improved formulations for the treatment of RB. We describe the current state of nanoparticles as a vehicle for gene delivery and cover the future prospects of this exciting area of ​​research. We also discuss the achievements, challenges and opportunities of nanomedicine to treat RB and mention new technical approaches that leverage our growing understanding of tumor biology and NP uptake mechanisms to design more effective nanotherapeutics for patients with RB.

Harmine (HM) is an alkaloid found in the seeds ofPagan HarmalaL., which has antimalarial and anticancer effects. However, its low bioavailability and toxic side effects have limited its clinical applications. This study used a novel mitochondrial targeting peptide (KA) to modify the HM-loaded liposome (KA-HM-LPS) by the pre-insertion method. The KA peptide consists of a mitochondrial targeting sequence (KLA) and a legume cleavable sequence (AAN). KA-HM-LPS had a small particle size of 246.2 ± 5.482 nm, a polydispersion index of 0.275 ± 0.038, a zeta potential of -15.5 ± 0.687 mV, and an encapsulation efficiency of 93.8% ± 0.96%. The drug release experiment showed that KA-HM-LPS was released more slowly than free HM in PBS buffer (pH 7.4). The kit-8 cell count assay and intracellular uptake experiments showed that KA-HM-LPS had greater cytotoxicity and mitochondrial targeting efficiency than HM-LPS in SK-Hep-1 overexpressing LGMN cells. The fluorescence observations of tetramethylrhodamine ethyl ester suggested that KA-HM-LPS had a reduced mitochondrial membrane potential. The tumor growth inhibition rate of KA-HM-LPS in vivo was as high as 67.74% ± 15.20%, far exceeding that of free HM-LPS (33.12% ± 8.96%). In addition, hematoxylin and eosin staining and blood compatibility assays showed that KA-HM-LPS had excellent biocompatibility and hemocompatibility. Therefore, KA-HM-LPS showed leguminous responsiveness and mitochondrial targeting and may represent a safe and efficient therapeutic strategy for hepatocellular carcinoma.

Due to rapid and significant advances in advanced materials and life sciences, nanotechnology is gaining increasing popularity. Among the numerous biomimetic transporters, inverse lyotropic liquid crystals are known for their unique properties. Due to their complicated topologies, these carriers allow the inclusion of molecules with different properties. Furthermore, the versatile symmetries of inverse LCNPs (lyotropic crystalline nanoparticles) and their aggregated phases allow their application in a variety of fields including drug delivery, food, cosmetics, materials science, etc. In this review, a detailed summary, discussion, and perspective on inverse lyotropic liquid crystals is provided.

Biomaterialien, Band 162, 2018, S. 47-59

Although extracellular barriers to siRNA delivery have been overcome through the use of advanced nanoparticle delivery systems, key intracellular barriers after internalization, including efficient siRNA degradation and endosomal escape, still remain a challenge. To address these issues, we designed a unique pH- and redox-responsive polyplex delivery system based on mPEG-b-PLA-PHis-ssPEI1.8k copolymer composed of a pH-responsive PEG-b -PLA-PHis copolymer ( Mw 5k) and a branched PEI (Mw 1.8k) linked to a redox cleavable disulfide bond. The copolymer showed excellent siRNA complexation and protection against endogenous substances at the relatively low N/P ratio of 6. The release of siRNA from the polyplexes (N/P 6) increased significantly from 13.62% to 58.67% under conditions simulating the endosomal microenvironment. The fluorescence resonance energy transfer (FRET) assay also showed a higher level of siRNA disassembly from the copolymer. Accelerated release of siRNA from polyplexes was significantly restricted when the N/P ratio was increased above 10 due to increased electrostatic interactions. Efficient endosomal escape of siRNA after internalization was confirmed by confocal microscopy, which was attributed to membrane destabilization of cleaved PEI strands, the “proton sponge effect” of PHis and PEI, and the relatively small size after disassembly. Increased disassembly and endosomal escape were elucidated as the main cause of polyplexes (N/P 6) showing more efficient Bcl-2 silencing (85.45%) than polyplexes with higher N/P ratios (N/P 10 and 15) . In vivo results further demonstrated that delivery of siBcl-2 by polyplexes (N/P 6) significantly inhibited MCF-7 breast tumor growth compared to its counterparts. Incorporation of non-electrical convertible interactions in equilibrium with electrostatic interactions in siRNA complexes proved to be an effective strategy to achieve efficient disassembly of stable polyplexes. Furthermore, polyplexes endowed with improved disassembly and endosomal escape offer a potential new way to address the intracellular delivery bottleneck for siRNA delivery.

Colloids and B Surfaces: Biointerfaces, Volume 188, 2020, Article 110804

Endosomal nanoparticle degradation is one of the main biological barriers associated with the drug delivery system. Nanoparticles are internalized into the cell via various endocytic pathways, where they are first delivered to early endosomes, which mature into the late endosome and lysosome. During this journey, the NP encounters a hostile chemical environment that leads to degradation of the NP and its contents. This process is collectively referred to as the intracellular defense against foreign substances. Therefore, to avoid this degrading fate, the endosomal escape technique has been studied for membrane fusion or membrane destabilization mechanisms. However, these methods are limited in application due to non-specific membrane fusion. To overcome this limitation, we designed a pH-responsive liposome composed of 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-liposome) in which only the cationic nitrogen and ammonium moiety occupies ~ 2.5% of the molecule. Such a small percentage of the cationic fraction is sufficient to exhibit pH responsive properties while maintaining DC liposome biocompatibility. DC-liposomes showed pH-dependent cationic properties due to protonation of the DC moiety at acidic pH. The fluorescence-based experiment confirmed the pH-dependent Fusogenic properties of DC liposomes. Furthermore, the endosomal colocalization study revealed a greater localization of DC-liposomes in the early endosome compared to the late endosome, suggesting a possible endosomal escape. Enhanced cationic and fusogenic properties of DC liposomes at acidic pH may mediate membrane fusion with the anionic endosomal membrane through electrostatic interaction, causing endosomal leakage. Furthermore, DC-liposome loaded with doxorubicin showed greater cytotoxicity than free doxorubicin, further supporting our endosomal escape. These results indicate the potential of DC liposomes to disrupt endosomal barriers to increase therapeutic efficacy, thereby guiding us in design considerations in the field of stimulus-responsive delivery devices.

International Journal of Pharmaceutics, Band 599, 2021, Artikel 120398

Antisense oligonucleotides (ASOs) are a new class of gene-specific therapies for diseases associated with the central nervous system (CNS). However, the transport of ASO across the blood-brain barrier (BBB) ​​to CNS target cells remains a major challenge. Since ASOs are taken up at the cerebral capillary endothelial cell interface primarily through endosomal pathways, entrapment in the endosomal compartment is a major obstacle to the efficient delivery of ASOs to the CNS. Therefore, we evaluated the efficacy of a panel of cell-penetrating peptides (CPPs) that target multiple endosomal escape domains for the intracellular delivery, endosomal delivery, and antisense activity of the FDA-approved spinraza (Nusinersen), an ASO for the treatment of muscle wasting. spinal cord (AME). We identified a CPP, HA2-ApoE(131–150), which, when conjugated with nusinersen, exhibited an efficient endosomal escape capability and significantly increased the level of complete functional survival of the motoneuron 2 mRNA (SMN2) in fibroblasts derived from SMA patients. Treatment ofSMN2Transgenic adult mice with this CPP-PMO conjugate resulted in a significant increase in complete contentSMN2in the brain and spinal cord. This work provides proof of principle that integration of endosomal escape domains with CPPs allows for enhanced cytosolic delivery of ASOs and, more importantly, improves the efficiency of BBB permeability and CNS activity of systemically administered ASOs.

Advanced Drug Delivery Reviews, Band 144, 2019, S. 90-111

The complexity of nanoscale interactions between biomaterials and cells has limited the realization of the ultimate vision of nanotechnology in diagnostics and therapy. Therefore, considerable effort has been expended to advance our understanding of the biophysical interactions of myriad nanoparticles. Endocytosis with nanomedicine has attracted great interest in the last decade. Here, we highlight the pervasive barriers to efficient intracellular delivery of nanoparticles and current advances and strategies employed to break through these barriers. We are also introducing new barriers that have been largely overlooked, such as the glycocalyx and macromolecular clumping. In addition, we draw attention to possible complications arising from disruption of the newly discovered functions of lysosomes. New strategies are being explored to exploit the intracellular defects inherent in disease states to improve the delivery and use of exosomes for bioanalytical and drug delivery. Additionally, we discuss advances in imaging techniques such as electron microscopy, high-resolution fluorescence microscopy, and single-particle tracking, which have significantly contributed to our growing understanding of intracellular pathways and nanoparticle transport. Finally, we endorse the push for a more intravital analysis of nanoparticle transport phenomena using the variety of techniques at our disposal. Unraveling the underlying mechanisms that govern cellular barriers to nanoparticle delivery and biological interactions will guide innovations that can break through these barriers.

European Journal of Pharmaceutics and Biopharmaceutics, Band 129, 2018, S. 184-190

In non-viral gene therapy, cationic polymers and lipids are commonly used to encapsulate macromolecular therapeutics in nanoparticles. Many biological barriers must be overcome on its journey to get the cargo to its intended intracellular destination. One of the main bottlenecks for efficient transfection is the endosomal barrier, as nanoparticles generally remain trapped in endosomes and are transported to lysosomes, where the cargo is broken down. The proton sponge hypothesis was introduced for cationic polymers in the late 1990s to explain their endosomal escape properties. However, due to many conflicting reports, no consensus on the validity of this hypothesis has been reached in the scientific community to date. Here we review the sometimes conflicting reports that have been published about the proton sponge hypothesis. We also discuss membrane destabilization and polymer swelling as additional factors that may affect endosomal escape from polyplexes. Based on the most important publications on the subject, we want to build a consensus on the role of the proton sponge hypothesis in endosomal escape.

Advanced Drug Delivery Reviews, Band 156, 2020, S. 119-132

Polymeric carriers are versatile tools for delivering therapeutic genes. Many polymers - when mounted on vehicles with nucleic acids - can protect cargo from degradation and releaseliveand facilitate its transport to intracellular compartments. Design options in polymer synthesis result in a comprehensive range of molecules and resulting vehicle formulations. These properties can be manipulated to achieve a stronger association with nucleic acid and cell loading, enhanced endosomal escape, or sustained delivery, depending on the application. Here we describe current approaches to using polymers and related strategies for gene delivery in preclinical and clinical applications. Polymeric vehicles that deliver genetic material have already reached important therapeutic parametersin vitroand in animal models. In our view, with pre-clinical trials that mimic those bestliveEnvironment, improved strategies for target specificity, and scalable techniques for polymer synthesis will further enhance the impact of this therapeutic approach.