Quality and Safety Considerations for Therapeutic Products Based on Extracellular Vesicles.

Extracellular vesicles (EVs) serve as an intrinsic system for delivering functional molecules within our body, playing significant roles in diverse physiological phenomena and diseases. Both native and engineered EVs are currently the subject of extensive research as promising therapeutics and drug delivery systems, primarily due to their remarkable attributes, such as targeting capabilities, biocompatibility, and low immunogenicity and mutagenicity. Nevertheless, their clinical application is still a long way off owing to multiple limitations. In this context, the Science Board of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan has conducted a comprehensive assessment to identify the current issues related to the quality and safety of EV-based therapeutic products. Furthermore, we have presented several examples of the state-of-the-art methodologies employed in EV manufacturing, along with guidelines for critical processes, such as production, purification, characterization, quality evaluation and control, safety assessment, and clinical development and evaluation of EV-based therapeutics. These endeavors aim to facilitate the clinical application of EVs and pave the way for their transformative impact in healthcare.

An approach for the intracellular delivery of IgG via enzymatic ligation with a cell-permeable attenuated cationic amphiphilic lytic peptide.

Achieving effective intracellular delivery of therapeutic molecules such as antibodies (IgG) is a challenge in biomedical research and pharmaceutical development. Conjugation of IgG with a cell-penetrating peptide is a rational approach. Here, not only the efficacy of the conjugates in internalizing into cells, but also the physicochemical property of the conjugates allowing their solubilized states in solution without forming aggregates are critical. In this study, we have shown that the first requirement can be addressed using a cell-permeable attenuated cationic amphiphilic lytic (CP-ACAL) peptide, L17ER4. The second requirement can be addressed by ligation of IgG to L17ER4 using sortase A, where the use of a linker of appropriate chain length is also important. For evaluation, the intracellular delivery efficacy was studied using conjugate structures with different orientations and conjugation modes of L17ER4 in ligation to a model protein, green fluorescent protein fused to a nuclear localization signal (NLS-EGFP). The effect of tetraarginine positioning in the L17ER4 sequence was also investigated. Following these studies, an optimized peptide sequence containing L17ER4 was ligated to an anti-green fluorescent protein (GFP) IgG bearing a sortase A recognition sequence. Treatment of the cells with the conjugate of anti-GFP IgG and L17ER4 resulted in a high efficiency of cytosolic translocation of the conjugate and the binding to the target protein in the cell without significant aggregate formation. The feasibility of the d-form of L17ER4 as a CP-ACAL was also confirmed.

Finding ways into the cytosol: Peptide-mediated approaches for delivering proteins into cells.

The delivery of functional proteins, including antibodies, into cells opens up many opportunities to regulate cellular events, with significant implications for studies in chemical biology and therapeutics. The inside of cells is isolated from the outside by the cell membrane. The hydrophilic nature of proteins prevents direct permeation of proteins through the cell membrane by passive diffusion. Therefore, delivery routes using endocytic uptake followed by endosomal escape have been explored. Alternatively, delivery concepts using transient permeabilization of cell membranes or effective promotion of endocytic uptake and endosomal escape using modified membrane-lytic peptides have been reported in recent years. Non-canonical protein delivery concepts, such as the use of liquid droplets or coacervates, have also been proposed. This review highlights some of the topics in peptide-mediated intracellular protein delivery.

Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.

ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

Light-controllable cell-membrane disturbance for intracellular delivery.

Highly polar and charged molecules, such as oligonucleotides, face significant barriers in crossing the cell membrane to access the cytoplasm. To address this problem, we developed a light-triggered twistable tetraphenylethene (TPE) derivative, TPE-C-N, to facilitate the intracellular delivery of charged molecules through an endocytosis-independent pathway. The central double bond of TPE in TPE-C-N is planar in the ground state but becomes twisted in the excited state. Under light irradiation, this planar-to-twisted structural change induces continuous cell membrane disturbances. Such disturbance does not lead to permanent damage to the cell membrane. TPE-C-N significantly enhanced the intracellular delivery of negatively charged molecules under light irradiation when endocytosis was inhibited through low-temperature treatment, confirming the endocytosis-independent nature of this delivery method. We have successfully demonstrated that the TPE-C-N-mediated light-controllable method can efficiently promote the intracellular delivery of charged molecules, such as peptides and oligonucleotides, with molecular weights ranging from 1000 to 5000 Da.

E3MPH16: An efficient endosomolytic peptide for intracellular protein delivery.

To facilitate the introduction of proteins, such as antibodies, into cells, a variety of delivery peptides have been engineered. These peptides are typically highly cationic and somewhat hydrophobic, enabling cytosolic protein delivery at the cost of causing cell damage by rupturing membranes. This balance between delivery effectiveness and cytotoxicity presents obstacles for their real-world use. To tackle this problem, we designed a new endosome-disruptive cytosolic delivery peptide, E3MPH16, inspired by mastoparan X (MP). E3MPH16 was engineered to incorporate three Glu (E3) and 16 His (H16) residues at the N- and C-termini of MP, respectively. The negative charges of E3 substantially mitigate the cell-surface damage induced by MP. The H16 segment is known to enhance cell-surface adsorption and endocytic uptake of the associated molecules. With these modifications, E3MPH16 was successfully trapped within endosomes. The acidification of endosomes is expected to protonate the side chains of E3 and H16, enabling E3MPH16 to rupture endosomal membranes. As a result, nearly 100% of cells achieved cytosolic delivery of a model biomacromolecule, Alexa Fluor 488-labeled dextran (10 kDa), via endosomal escape by co-incubation with E3MPH16. The delivery process also suggested the involvement of macropinocytosis and caveolae-mediated endocytosis. With the assistance of E3MPH16, Cre recombinase and anti-Ras-IgG delivered into HEK293 cells and HT1080 cells enabled gene recombination and inhibited cell proliferation, respectively. The potential for in vivo application of this intracellular delivery method was further validated by topically injecting the green fluorescent protein fused with a nuclear localization signal (NLS-GFP) along with E3MPH16 into Colon-26 tumor xenografts in mice.

Liquid Droplet-Mediated Formulation of Lipid Nanoparticles Encapsulating Immunoglobulin G for Cytosolic Delivery.

Antibodies are promising biopharmaceuticals that offer new therapeutic options for diseases. Since antibodies are membrane impermeable, approaches that allow immunoglobulin Gs (IgGs) to access intracellular therapeutic targets would open new horizons in antibody therapies. Lipid nanoparticles (LNPs) are among the classes of vectors that deliver biopharmaceuticals into cells. Using liquid droplets formed by IgG and polyglutamate, we report here a unique approach to forming LNPs containing IgG via liquid droplets formed in the presence of polyglutamic acid (polyE). The addition of polyE promoted the formation of smaller LNPs with cationic lipids than in its absence, and the formed LNPs were much more efficient in cytosolic IgG delivery and targeting of cellular proteins. This approach also allows for the encapsulation of intact IgG without the need for chemical or sequence modification. The intracellularly delivered IgG retained its target binding ability, as demonstrated by labeling of nuclear pore complex and HRas-GFP and inhibition of antiapoptotic cell death by phosphorylated Akt protein in live cells.