Intracellular Delivery Vehicles Research Articles

Effect of Hydroxyapatite Nanoparticle Crystallinity and Colloidal Stability on Cytotoxicity.

Hydroxyapatite nanoparticles (nHA) have gained attention as potential intracellular drug delivery vehicles due to their high binding affinity for various biomolecules and pH-dependent solubility. Yet, the dependence of nHA cytocompatibility on their physicochemical properties remains unclear since numerous studies have revealed starkly contrasting results. These discrepancies may be attributed to differences in size, shape, crystallinity, and aggregation state of nHA, which complicates fundamental understanding of the factors driving nHA cytotoxicity. Here, we hypothesize that nHA cytotoxicity is primarily driven by intracellular calcium levels following the internalization of nHA nanoparticles. By investigating the cytotoxicity of spherical nHA with different crystallinity and dispersity, we find that both lower crystallinity and increased agglomeration of nHA raise cytotoxicity, with nanoparticle agglomeration being the more dominant factor. We show that the internalization of nHA enhances intracellular calcium levels and increases the production of reactive oxygen species (ROS). However, only subtle changes in intracellular calcium are observed, and their physiological relevance remains to be confirmed. In conclusion, we show that nHA agglomeration enhances ROS production and the associated cytotoxicity. These findings provide important guidelines for the future design of nHA-containing formulations for biomedical applications, implying that nHA crystallinity and especially agglomeration should be carefully controlled to optimize biocompatibility and therapeutic efficacy.

Quinine-Based Polymers Are Versatile and Effective Vehicles for Intracellular pDNA, mRNA, and Cas9 Protein Delivery.

Quinine-based polymers have previously demonstrated promising performance in delivering pDNA in cells owing to their electrostatic as well as the nonelectrostatic interactions with pDNA. Herein, we evaluate whether quinine-based polymers are versatile for delivery of mRNA and Cas9-sgRNA complexes, especially in a serum-rich environment. Both mRNA and the Cas9-sgRNA complex are potent therapeutics that are structurally, chemically, and functionally very different from pDNA. By exploring a family of 7 quinine-based polymers that vary in monomer structure and polymer composition, we tested numerous formulations (42 with pDNA, 96 with mRNA, and 48 with Cas9-sgRNA) for payload-polymer complexation and delivery to compare payload-dependent structure-activity relationships. Several formulations demonstrated performance comparable to or better than the commercially available transfection agent jetPEI. The results of this study demonstrate the potential of quinine-based as a versatile carrier platform for delivering a wide range of nucleic acid therapeutics and serving the drug delivery needs in the field genetic medicine.

Cellular Uptake of Phase-Separating Peptide Coacervates.

Peptide coacervates self-assembling via liquid-liquid phase separation are appealing intracellular delivery vehicles of macromolecular therapeutics (proteins, DNA, mRNA) owing to their non-cytotoxicity, high encapsulation capacity, and efficient cellular uptake. However, the mechanisms by which these viscoelastic droplets cross the cellular membranes remain unknown. Here, using multimodal imaging, data analytics, and biochemical inhibition assays, identify the key steps by which droplets enter the cell. find that the uptake follows a non-canonical pathway and instead integrates essential features of macropinocytosis and phagocytosis, namely active remodeling of the actin cytoskeleton and appearance of filopodia-like protrusions. Experiments using giant unilamellar vesicles show that the coacervates attach to the bounding membrane in a charge- and cholesterol-dependent manner but do not breach the lipid bilayer barrier. Cell uptake in the presence of small molecule inhibitors - interfering with actin and tubulin polymerization - confirm the active role of cytoskeleton remodeling, most prominently evident in electron microscopy imaging. These findings suggest a peculiar internalization mechanism for viscoelastic, glassy coacervate droplets combining features of non-specific uptake of fluids by macropinocytosis and particulate uptake of phagocytosis. The broad implications of this study will enable to enhance the efficacy and utility of coacervate-based strategies for intracellular delivery of macromolecular therapeutics.

Improvement of Therapeutic Effect via Inducing Non-Apoptotic Cell Death Using mRNA-Protection Nanocage.

Necroptosis, a cell death mechanism with the characteristics of both apoptosis and necrosis, is proposed as a promising therapeutic approach for cancer therapy. Induction of necroptosis for cancer therapy may be possible through the regulation of the expression of a key factor gene receptor-interacting protein kinase-3 (RIPK3) via in vitro transcription (IVT) mRNA delivery. However, mRNA is susceptible to degradation and has a low delivery efficiency, which highlights the requirement of a proper delivery vehicle for intracellular delivery. Therefore, a new mRNA delivery system based on the nanostructured silica nanoparticles, termed mRNA-protective nanocage (mPN) has been developed. High-efficiency expression of RIPK3 and induction of necroptosis is achieved through delivery of RIPK3 IVT mRNA with mPN in vitro and in vivo models. Importantly, the mPN carrying RIPK3 mRNA distributed locally in tumors upon intravascular injection, and successfully induced necroptosis and immune cell infiltration, a hallmark of necroptosis. the suppression of tumor growth in a murine cancer model, demonstrating the synergistic effect of RIPK3 mRNA- and immune cell-mediated therapy is also observed. These findings suggest the potential for anticancer therapy through necroptosis induction and provide a strategy for the development of mRNA-based nanomedicine.

Injectable Mesocellular Foam Silica Microparticles with a Dual Role of Cell‐Recruiting Scaffolds and Intracellular Delivery Vehicles for Enhanced Cancer Vaccine

AbstractThe cancer vaccine is one of the potent cancer immunotherapies based on provoking antigen‐specific adaptive immune responses in patients. Activating host antigen‐presenting cells by delivering antigens and adjuvants is one of the most important requirements to enhance cancer vaccine efficacy. This study proposes an effective cancer vaccine platform based on injectable mesocellular foam (MCF) silica microparticles with a dual role capable of in situ recruitment of host dendritic cells (DCs) and intracellular delivery of antigens to the recruited DCs. Subcutaneously administrated MCF microparticles loaded with chemoattractant to DCs (GM‐CSF), antigenic protein (OVA), and TLR9 agonist (CpG‐ODN) lead to robust DC maturation and high antigen‐specific T cell responses. The cell recruitment, DC activation, and T cell responses of MCF‐based cancer vaccine are significantly higher than injectable mesoporous silica scaffold, of which role is mostly limited to the cell recruiting scaffold. These features of MCF lead to effective inhibition of tumor growth in prophylactic vaccine setting and lung metastatic cancer model, representing MCF enabling cell‐recruiting scaffold formation and intracellular delivery of antigen can be a promising material platform for an enhanced cancer vaccine.

A Self-Immolative DNA Nanogel Vaccine toward Cancer Immunotherapy.

The development of precisely engineered vehicles for intracellular delivery and the controlled release of payloads remains a challenge. DNA-based nanomaterials offer a promising solution based on the A-T-G-C alphabet-dictated predictable assembly and high programmability. Herein, we present a self-immolative DNA nanogel vaccine, which can be tracelessly released in the intracellular compartments and activate the immune response. Three building blocks with cytosine-rich overhang domains are designed to self-assemble into a DNA nanogel framework with a controlled size. Two oligo agonists and one antigen peptide are conjugated to the building blocks via an acid-labile chemical linker. Upon internalization into acidic endosomes, the formation of i-motif configurations leads to dissociation of the DNA nanogel vaccine. The acid-labile chemical linker is cleaved, releasing the agonists and antigen in their traceless original form to activate antigen-presenting cells and an immune response. This study presents a novel strategy for constructing delivery platforms for intracellularly stimuli-triggered traceless release of therapeutics.

Photo‐Responsive Phase‐Separating Fluorescent Molecules for Intracellular Protein Delivery

AbstractCellular membranes, including the plasma and endosome membranes, are barriers to outside proteins. Various vehicles have been devised to deliver proteins across the plasma membrane, but in many cases, the payload gets trapped in the endosome. Here we designed a photo‐responsive phase‐separating fluorescent molecule (PPFM) with a molecular weight of 666.8 daltons. The PPFM compound condensates as fluorescent droplets in the aqueous solution by liquid‐liquid phase separation (LLPS), which disintegrate upon photoirradiation with a 405 nm light‐emitting diode (LED) lamp within 20 min or a 405 nm laser within 3 min. The PPFM coacervates recruit a wide range of peptides and proteins and deliver them into mammalian cells. Photolysis disperses the payload from condensates into the cytosolic space. Altogether, a type of small molecules that are photo‐responsive and phase separating are discovered; their coacervates can serve as transmembrane vehicles for intracellular delivery of proteins, whereas photo illumination triggers the cytosolic distribution of the payload.

Coacervate droplets: Emerging vehicles for intracellular delivery

Coacervate droplets: Emerging vehicles for intracellular delivery

Design, preparation and characterization of octopus-like self-releasing intracellular protein transporter LEB5 based on Escherichia coli heat-labile enterotoxin

Despite the great potential of protein drugs as intracellular therapeutic agents, the unmet challenge in breaking through the cell membrane barrier and delivering them to intracellular targets remains. Therefore, developing safe and effective delivery vehicles is critical for fundamental biomedical research and clinical applications. In this study, we designed an octopus-like self-releasing intracellular protein transporter, the LEB5, based on the heat-labile enterotoxin. This carrier comprises five identical units, each of which has three main components: a linker, a self-releasing enzyme sensitivity loop, and the LTB transport domain. The LEB5 comprises five purified monomers that self-assemble to create a pentamer with ganglioside GM1 binding capacity. The fluorescent protein EGFP was used as a reporter system to identify the LEB5 features. The high-purity fusion protein ELEB monomer was produced from modified bacteria carrying pET24a(+)-eleb recombinant plasmids. EGFP protein could effectively detach from LEB5 by low dosage trypsin, according to electrophoresis analysis. The transmission electron microscopy results indicate that both LEB5 and ELEB5 pentamers exhibit a relatively regularly spherical shape, and the differential scanning calorimetry measurements further suggest that these proteins possess excellent thermal stability. Fluorescence microscopy revealed that LEB5 translocated EGFP into different cell types. Flow cytometry showed cellular differences in the transport capacity of LEB5. According to the confocal microscopy, fluorescence analysis and western blotting data, EGFP was transferred to the endoplasmic reticulum by the LEB5 carrier, detached from LEB5 by cleavage of the enzyme-sensitive loop, and released into the cytoplasm. Within the dosage range of LEB5 10–80 μg/mL, cell counting kit-8 assay revealed no significant changes in cell viability. These results demonstrated that LEB5 is a safe and effective intracellular self-releasing delivery vehicle capable of transporting and releasing protein medicines into cells.

Quantitative Analysis of Extracellular Vesicle Uptake and Fusion with Recipient Cells.

In precision medicine, extracellular vesicles (EVs) are promising intracellular drug delivery vehicles. The development of a quantitative analysis approach will provide valuable information from the perspective of cell biology and system design for drug delivery. Previous studies have reported quantitative methods to analyze the relative uptake or fusion of EVs to recipient cells. However, relatively few methods have enabled the simultaneous evaluation of the "number" of EVs taken up by recipient cells and those that fuse with cellular membranes. In this study, we report a simple quantitative method based on the NanoBiT system to quantify the uptake and fusion of small and large EVs (sEVs and lEVs, respectively). We assessed the abundance of these two subtypes of EVs and determined that lEVs may be more effective vehicles for transporting cargo to recipient cells. The results also indicated that both sEVs and lEVs have very low fusogenic activity, which can be improved in the presence of a fusogenic protein.

Adenovirus Fibers as Ultra-Stable Vehicles for Intracellular Nanoparticle and Protein Delivery.

Protein-based carriers are promising vehicles for the intracellular delivery of therapeutics. In this study, we designed and studied adenovirus protein fiber constructs with potential applications as carriers for the delivery of protein and nanoparticle cargoes. We used as a basic structural framework the fibrous shaft segment of the adenovirus fiber protein comprising of residues 61–392, connected to the fibritin foldon trimerization motif at the C-terminal end. A fourteen-amino-acid biotinylation sequence was inserted immediately after the N-terminal, His-tagged end of the construct in order to enable the attachment of a biotin moiety in vivo. We report herein that this His-tag biotinylated construct folds into thermally and protease-stable fibrous nanorods that can be internalized into cells and are not cytotoxic. Moreover, they can bind to proteins and nanoparticles through the biotin–streptavidin interaction and mediate their delivery to cells. We demonstrate that streptavidin-conjugated gold nanoparticles can be transported into NIH3T3 fibroblast and HeLa cancer cell lines. Furthermore, two streptavidin-conjugated model proteins, alkaline phosphatase and horseradish peroxidase can be delivered into the cell cytoplasm in their enzymatically active form. This work is aimed at establishing the proof-of-principle for the rational engineering of diverse functionalities onto the initial protein structural framework and the use of adenovirus fiber-based proteins as nanorods for the delivery of nanoparticles and model proteins. These constructs could constitute a stepping stone for the development of multifunctional and modular fibrous nanorod platforms that can be tailored to applications at the sequence level.

Cyclic peptides nanospheres: A ‘2-in-1′ self-assembled delivery system for targeting nucleus and cytoplasm

Vascular endothelial growth factor (VEGF) is considered as one of the vital growth factors for angiogenesis, which is primarily responsible for the progress and maintenance of new vascular network in tumor. Numerous studies report that inhibition of VEGF-induced angiogenesis is a potent technique for cancer suppression. Recently, RNA interference, especially small interfering RNA (siRNA) signified a promising approach to suppress the gene expression. However, the clinical implementation of biological macromolecules such as siRNA is significantly limited because of stability and bioavailability issues. Herein, self-assembled peptide nanospheres have been generated from L,L-cyclic peptides using hydrophobic (Trp), positively charged (Arg) and cysteine (Cys) amino acid residues and demonstrated as vehicles for intracellular delivery of VEGF siRNA and VEGF antisense oligonucleotide. Formation of peptide nanostructures is confirmed by HR-TEM, AFM, SEM and DLS analysis. Possible mechanism of self-assembly of the cyclic peptides and their binding with macromolecules are demonstrated by in-silico analysis. Gel electrophoresis reveals that the newly generated peptide based organic materials exhibit strong binding affinity toward siRNAs / antisense oligonucleotides (ASOs) at optimum concentration. Flow cytometry and confocal microscopy results confirm the efficiency of the new biomaterials toward the intracellular delivery of fluorescent labeled siRNA / ASOs. Furthermore, VEGF expression evaluated by western blot and RT-PCR upon the delivery of functional VEGF siRNA/ASOs suggests that very low concentrations of VEGF siRNA/ASOs cause significant gene knockdown at protein and mRNA levels, respectively.

Recent advances in the intracellular delivery of macromolecule therapeutics.

Intracellular delivery of macromolecules is a critical procedure for biological research and drug discovery, including proteins, peptides, vaccines, antibodies and genes. The penetration of macromolecule therapeutics through the cell membrane to intracellular targets is a prerequisite for their biological activity, but most delivery systems rely on the endocytic pathway to enter the cell and confront an inability to escape from the lysosome. A profound understanding of the cellular internalization of transporting carriers can (i) optimize the design of drug delivery systems, (ii) maintain the biological activity of biomolecular drugs, (iii) improve the efficiency of intracellular macromolecule transport and release, (iv) bring new opportunities for the discovery of macromolecule therapeutics and treatment of refractory disease. This article summarizes the uptake pathway of intracellular delivery vehicles for macromolecule drugs, hoping to provide ideas and references for macromolecule therapeutics delivery systems.

Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer.

CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant Kras G12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.

Uncoupling the Folding-Function Paradigm of Lytic Peptides to Deliver Impermeable Inhibitors of Intracellular Protein-Protein Interactions.

Here, we describe the use of peptide backbone N-methylation as a new strategy to transform membrane-lytic peptides (MLPs) into cytocompatible intracellular delivery vehicles. The ability of lytic peptides to engage with cell membranes has been exploited for drug delivery to carry impermeable cargo into cells, but their inherent toxicity results in narrow therapeutic windows that limit their clinical translation. For most linear MLPs, a prerequisite for membrane activity is their folding at cell surfaces. Modification of their backbone with N-methyl amides inhibits folding, which directly correlates to a reduction in lytic potential but only minimally affects cell entry. We synthesized a library of N-methylated peptides derived from MLPs and conducted structure-activity studies that demonstrated the broad utility of this approach across different secondary structures, including both β-sheet and helix-forming peptides. Our strategy is highlighted by the delivery of a notoriously difficult class of protein-protein interaction inhibitors that displayed on-target activity within cells.

Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications.

Synthetic cells have a major role in gaining insight into the complex biological processes of living cells; they also give rise to a range of emerging applications from gene delivery to enzymatic nanoreactors. Living cells rely on compartmentalization to orchestrate reaction networks for specialized and coordinated functions. Principally, the compartmentalization has been an essential engineering theme in constructing cell-mimicking systems. Here, efforts to engineer liquid-liquid interfaces of multiphase systems into membrane-bounded and membraneless compartments, which include lipid vesicles, polymer vesicles, colloidosomes, hybrids, and coacervate droplets, are summarized. Examples are provided of how these compartments are designed to imitate biological behaviors or machinery, including molecule trafficking, growth, fusion, energy conversion, intercellular communication, and adaptivity. Subsequently, the state-of-art applications of these cell-inspired synthetic compartments are discussed. Apart from being simplified and cell models for bridging the gap between nonliving matter and cellular life, synthetic compartments also are utilized as intracellular delivery vehicles for nuclei acids and nanoreactors for biochemical synthesis. Finally, key challenges and future directions for achieving the full potential of synthetic cells are highlighted.

Toxin-Mediated siRNA Delivery

AB toxins with built-in cell targeting and endosomal escape mechanisms are attractive intracellular delivery vehicles. However, their compatibility with nucleic-acid-based therapeutics is not fully explored. Arnold et al. demonstrated the first functional siRNA delivery by diphtheria toxin (DT) in vitro, marking an important step in expanding the utility of AB toxins for nucleic acid delivery.

Synthesis of Poly(acyclic orthoester)s: Acid‐Sensitive Biomaterials for Enhancing Immune Responses of Protein Vaccine

AbstractWhile poly(acyclic orthoester)s (PAOEs) have many appealing features for drug delivery, their application is significantly hindered by a lack of facile synthetic methods. Reported here is a simple method for synthesizing acyclic diketene acetal monomers from diols and vinyl ether, and their polymerization with a diol to first synthesize PAOEs. The PAOEs rapidly hydrolyze at lysosomal pH. With the help of a cationic lipid, ovalbumin, a model vaccine antigen was efficiently loaded into PAOEs nanoparticles using a double emulsion method. These nanoparticles efficiently delivered ovalbumin into the cytosol of dendritic cells and demonstrated enhanced antigen presentation over poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles. PAOEs are promising vehicles for intracellular delivery of biopharmaceuticals and could increase the utility of poly(orthoesters) in biomedical research.

Synthesis of Poly(acyclic orthoester)s: Acid-Sensitive Biomaterials for Enhancing Immune Responses of Protein Vaccine.

While poly(acyclic orthoester)s (PAOEs) have many appealing features for drug delivery, their application is significantly hindered by a lack of facile synthetic methods. Reported here is a simple method for synthesizing acyclic diketene acetal monomers from diols and vinyl ether, and their polymerization with a diol to first synthesize PAOEs. The PAOEs rapidly hydrolyze at lysosomal pH. With the help of a cationic lipid, ovalbumin, a model vaccine antigen was efficiently loaded into PAOEs nanoparticles using a double emulsion method. These nanoparticles efficiently delivered ovalbumin into the cytosol of dendritic cells and demonstrated enhanced antigen presentation over poly(lactic-co-glycolic acid) (PLGA) nanoparticles. PAOEs are promising vehicles for intracellular delivery of biopharmaceuticals and could increase the utility of poly(orthoesters) in biomedical research.

Modular design and self-assembly of multidomain peptides towards cytocompatible supramolecular cell penetrating nanofibers.

The discovery of cell penetrating peptides (CPPs) with unique membrane activity has inspired the design and synthesis of a variety of cell penetrating macromolecules, which offer tremendous opportunity and promise for intracellular delivery of a variety of imaging probes and therapeutics. While cell penetrating macromolecules can be designed and synthesized to have equivalent or even superior cell penetrating activity compared with natural CPPs, most of them suffer from moderate to severe cytotoxicity. Inspired by recent advances in peptide self-assembly and cell penetrating macromolecules, in this work, we demonstrated a new class of peptide assemblies with intrinsic cell penetrating activity and excellent cytocompatibility. Supramolecular assemblies were formed through the self-assembly of de novo designed multidomain peptides (MDPs) with a general sequence of Kx(QW)6Ey in which the numbers of lysine and glutamic acid can be varied to control supramolecular assembly, morphology and cell penetrating activity. Both supramolecular spherical particles and nanofibers exhibit much higher cell penetrating activity than monomeric MDPs while supramolecular nanofibers were found to further enhance the cell penetrating activity of MDPs. In vitro cell uptake results suggested that the supramolecular cell penetrating nanofibers undergo macropinocytosis-mediated internalization and they are capable of escaping from the lysosome to reach the cytoplasm, which highlights their great potential as highly effective intracellular therapeutic delivery vehicles and imaging probes.