Antibody-Free Glycogen Nanoparticles Engage Human Immune T Cells for Intracellular Delivery of Small Drugs or mRNA.

T cells play a major role in immune defense against viral infections and diseases such as cancer. Accordingly, developing nanoparticle (NP) systems to effectively deliver therapeutics to T cells is of interest. However, NP-mediated delivery of drugs to T cells is challenging because of the nonphagocytic nature of T cells. To engage T cells and induce cellular internalization, NPs are typically decorated with specific receptor-targeting antibodies, often using laborious and costly procedures. Herein, we report that natural glycogen NPs (i.e., nanosugars) with different sizes (20-80 nm) and surface charges (neutral and positively charged) engage Jurkat T cells, undergo intracellular trafficking, and release encapsulated drug without the use of receptor-targeting antibodies. Specifically, glycogen-resveratrol constructs are employed to reactivate HIV-1 latently infected Jurkat T cells (J-Lat A2) and trigger proviral expression. Both neutral and positively charged glycogen NPs engage with J-Lat A2 cells. Large (84 ± 29 nm) and positively charged (23 ± 5 mV) NPs, denoted phytoglycogen-ethylenediamine (PGEDA) NPs, readily associate with the cell membrane and are internalized (60%) in J-Lat A2 cells but remain confined in the endocytic vesicles, with moderate reactivation of latent HIV-1 (4.7 ± 0.5%). Conversely, small (21 ± 5 nm) and positively charged (10 ± 6 mV) NPs, bovine glycogen-EDA (BGEDA) NPs, associate slowly with T cells but show nearly 100% internalization and efficient endosomal escape properties, resulting in 1.5-fold higher reactivation of latent HIV-1 in T cells. PGEDA NPs and BGEDA NPs are also internalized by primary human T cells (>90% cell association) and enable the transfection of mRNA, with BGEDA NPs showing a 2-fold higher transfection than PGEDA NPs. This work highlights the potential of BGEDA NPs for the effective intracellular delivery of small-molecule drugs and mRNA in T cells.

Efficient delivery of curcumin by functional solid lipid nanoparticles with promoting endosomal escape and liver targeting properties.

In the realm of intracellular drug delivery, overcoming the barrier of endosomal entrapment stands as a critical factor influencing the effectiveness of nanodrug delivery systems. This study focuses on the synthesis of an acid-sensitive fatty acid derivative called imidazole-stearic acid (IM-SA). Leveraging the proton sponge effect attributed to imidazole groups, IM-SA was anticipated to play a pivotal role in facilitating endosomal escape. Integrated into the lipid core of solid lipid nanoparticles (SLNs), IM-SA was paired with hyaluronic acid (HA) coating on the surface of SLNs loading with curcumin (CUR). The presence of IM-SA and HA endowed HA-IM-SLNs@CUR with dual functionalities, enabling the promotion of endosomal escape, and specifical targeting of liver cancer. HA-IM-SLNs@CUR exhibited a particle size of ∼228 nm, with impressive encapsulation efficiencies (EE) of 87.5 % ± 2.3 % for CUR. Drugs exhibit significant pH sensitive release behavior. Cellular experiments showed that HA-IM-SLN@CUR exhibits enhanced drug delivery capability. The incorporation of IM-SA significantly improved the endosomal escape of HA-IM-SLN@CUR, facilitating accelerated intracellular drug release and increasing intracellular drug concentration, exhibiting excellent growth inhibitory effects on HepG2 cells. Animal experiments revealed a 3.4-fold increase in CUR uptake at the tumor site with HA-IM-SLNs@CUR over the free CUR, demonstrating remarkable tumor homing potential with the tumor growth inhibition rate of 97.2 %. These findings indicated the significant promise of HA-IM-SLNs@CUR in the realm of cancer drug delivery.

Novel targeting liposomes with enhanced endosomal escape for co-delivery of doxorubicin and curcumin.

Effective endosomal escape is crucial for enhancing the efficiency of nanodrug delivery systems. In this study, we developed a novel liposomal system utilizing acid-sensitive N-(3-amino-propyl) imidazole cholesterol (IM-Chol), specifically designed for the targeted co-delivery of doxorubicin (DOX) and curcumin (CUR) to hepatocellular carcinoma (HCC). Designated as GA-IM-LIP@DOX/CUR, this liposomal system incorporates glycyrrhetinic acid (GA) to improve target specificity toward HCC cells. Notably, both drugs exhibited pH-sensitive release profiles, facilitating precise drug release within acidic environments. Our investigation into cellular uptake demonstrated that modified liposomes, GA-IM-LIP@FITC and IM-LIP@FITC, achieved progressively enhanced intracellular accumulation of FITC compared to unmodified liposomes. Competitive inhibition assays utilizing free GA further validated the targeting efficacy of GA. Moreover, the GA-IM-LIP@FITC and IM-LIP@FITC groups exhibited rapid endosomal escape of FITC within the first two hours, in contrast to delayed escape observed in the LIP@FITC group, confirming that the protonation of IM-Chol promotes drug release into the cytosol. In vivo studies substantiated that GA-IM-LIP@DOX/CUR effectively inhibited tumor growth. This research provides significant insights into the design and functionality of the GA-IM-LIP@DOX/CUR liposomal system, underscoring its potential to enhance drug delivery strategies in the treatment of HCC.

Azobenzene-based liposomes with nanomechanical action for cytosolic chemotherapeutic drug delivery.

The stimuli-responsive nano-carriers are at the forefront of research in nanotechnology and materials science. These advanced systems are designed to alter their physicochemical properties upon exposure to specific stimuli, enabling controllable and targeted delivery of therapeutic agents. Nevertheless, limited endosomal escape reduces the drug bioavailability in clinical use. We herein report azobenzene (Azo)-based liposomes, prepared by co-assembling the photoisomerizable cationic Azo lipids and helper lipids, which achieve controllable doxorubicin (Dox) release and enhanced cytosolic transport upon light irradiation. Azo lipids undergo reversible isomerization between cis-isomers and trans-isomer when received UV and visible (Vis) light irradiation, causing liposomal membrane permeability changes for controlled drug release. Moreover, the nanomechanical action created by the isomerization of Azo lipids promotes the endosomal escape of the liposomes. DSPC-Azo liposomes, with minimal Dox leakage, showed significant tumor cell killing upon irradiation. For in vivo study, we co-encapsulated the upconverting nanoparticles (UCNPs), which can convert the near-infrared (NIR) light into UV/Vis emissions, facilitating Azo units activation. UCNP/Dox-loaded DSPC-Azo liposomes inhibited tumor growth under NIR irradiation in a 4T1 tumor-bearing mouse model.

Broadening the Scope of Sapofection: Cationic Peptide-Saponin Conjugates Improve Gene Delivery In Vitro and In Vivo.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Multicompartment Polyion Complex Micelles Based on Triblock Polypept(o)ides Mediate Efficient siRNA Delivery to Cancer-Associated Fibroblasts for Antistromal Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and the third leading cause for cancer-related death worldwide. The tumor is difficult-to-treat due to its inherent resistance to chemotherapy. Antistromal therapy is a novel therapeutic approach, targeting cancer-associated fibroblasts (CAF) in the tumor microenvironment. CAF-derived microfibrillar-associated protein 5 (MFAP-5) is identified as a novel target for antistromal therapy of HCC with high translational relevance. Biocompatible polypept(o)ide-based polyion complex micelles (PICMs) constructed with a triblock copolymer composed of a cationic poly(l-lysine) complexing anti-MFAP-5 siRNA (siMFAP-5) via electrostatic interaction, a poly(γ-benzyl-l-glutamate) block loading cationic amphiphilic drug desloratatine (DES) via π-π interaction as endosomal escape enhancer and polysarcosine poly(N-methylglycine) for introducing stealth properties, are generated for siRNA delivery. Intravenous injection of siMFAP-5/DES PICMs significantly reduces the hepatic tumor burden in a syngeneic implantation model of HCC, with a superior MFAP-5 knockdown effect over siMFAP-5 PICMs or lipid nanoparticles. Transcriptome and histological analysis reveal that MFAP-5 knockdown inhibited CAF-related tumor vascularization, suggesting the anti-angiogenic effect of RNA interference therapy. In conclusion, multicompartment PICMs combining siMFAP-5 and DES in a single polypept(o)ide micelle induce a specific knockdown of MFAP-5 and demonstrate a potent antitumor efficacy (80% reduced tumor burden vs untreated control) in a clinically relevant HCC model.

Rational Design of Lipid-Based Vectors for Advanced Therapeutic Vaccines.

Recent advancements in vaccine delivery systems have seen the utilization of various materials, including lipids, polymers, peptides, metals, and inorganic substances, for constructing non-viral vectors. Among these, lipid-based nanoparticles, composed of natural, synthetic, or physiological lipid/phospholipid materials, offer significant advantages such as biocompatibility, biodegradability, and safety, making them ideal for vaccine delivery. These lipid-based vectors can protect encapsulated antigens and/or mRNA from degradation, precisely tune chemical and physical properties to mimic viruses, facilitate targeted delivery to specific immune cells, and enable efficient endosomal escape for robust immune activation. Notably, lipid-based vaccines, exemplified by those developed by BioNTech/Pfizer and Moderna against COVID-19, have gained approval for human use. This review highlights rational design strategies for vaccine delivery, emphasizing lymphoid organ targeting and effective endosomal escape. It also discusses the importance of rational formulation design and structure-activity relationships, along with reviewing components and potential applications of lipid-based vectors. Additionally, it addresses current challenges and future prospects in translating lipid-based vaccine therapies for cancer and infectious diseases into clinical practice.

Dual Effect by Chemical Electron Transfer Enhanced siRNA Lipid Nanoparticles: Reactive Oxygen Species-Triggered Tumor Cell Killing Aggravated by Nrf2 Gene Silencing.

Insufficient endosomal escape presents a major hurdle for successful nucleic acid therapy. Here, for the first time, a chemical electron transfer (CET) system was integrated into small interfering RNA (siRNA) lipid nanoparticles (LNPs). The CET acceptor can be chemically excited using the generated energy between the donor and hydrogen peroxide, which triggers the generation of reactive oxygen species (ROS), promoting endosomal lipid membrane destabilization. Tetra-oleoyl tri-lysino succinoyl tetraethylene pentamine was included as an ionizable lipopeptide with a U-shaped topology for effective siRNA encapsulation and pH-induced endosomal escape. LNPs loaded with siRNA and CET components demonstrated a more efficient endosomal escape, as evidenced by a galectin-8-mRuby reporter; ROS significantly augmented galectin-8 recruitment by at least threefold compared with the control groups, with a p value of 0.03. Moreover, CET-enhanced LNPs achieved a 24% improvement in apoptosis level by knocking down the tumor-protective gene nuclear factor erythroid 2-related factor 2, boosting the CET-mediated ROS cell killing.

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.

Local delivery of accutox® synergises with immune-checkpoint inhibitors at disrupting tumor growth.

BACKGROUND: The Accum® platform was initially designed to accumulate biomedicines in target cells by inducing endosomal-to-cytosol escape. Interestingly however, the use of unconjugated Accum® was observed to trigger cell death in a variety of cancer cell lines; a property further exploited in the development of Accum®-based anti-cancer therapies. Despite the impressive pro-killing abilities of the parent molecule, some cancer cell lines exhibited resistance. This prompted us to test additional Accum® variants, which led to the identification of the AccuTOX® molecule. METHODS: A series of flow-cytometry and cell-based assays were used to assess the pro-killing properties of AccuTOX® along with its ability to trigger the production of reactive oxygen species (ROS), endosomal breaks and antigen presentation. RNA-seq was also conducted to pinpoint the most prominent processes modulated by AccuTOX® treatment in EL4 T-cell lymphoma. Finally, the therapeutic potency of intratumorally-injected AccuTOX® was evaluated in three different murine solid tumor models (EL4, E0771 and B16) both as a monotherapy or in combination with three immune-checkpoint inhibitors (ICI). RESULTS: In total, 7 Accum® variants were screened for their ability to induce complete cell death in 3 murine (EL4, B16 and E0771) and 3 human (MBA-MD-468, A549, and H460) cancer cell lines of different origins. The selected compound (hereafter refereed to as AccuTOX®) displayed an improved killing efficiency (~ 5.5 fold compared to the parental Accum®), while retaining its ability to trigger immunogenic cell death, ROS production, and endosomal breaks. Moreover, transcriptomic analysis revealed that low dose AccuTOX® enhances H2-Kb cell surface expression as well as antigen presentation in cancer cells. The net outcome culminates in impaired T-cell lymphoma, breast cancer and melanoma growth in vivo especially when combined with anti-CD47, anti-CTLA-4 or anti-PD-1 depending on the animal model. CONCLUSIONS: AccuTOX® exhibits enhanced cancer killing properties, retains all the innate characteristics displayed by the parental Accum® molecule, and synergizes with various ICI in controlling tumor growth. These observations will certainly pave the path to continue the clinical development of this lead compound against multiple solid tumor indications.

A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

Selective targeting of chemically modified miR-34a to prostate cancer using a small molecule ligand and an endosomal escape agent.

Use of tumor-suppressive microRNAs (miRNAs) as anti-cancer agents is hindered by the lack of effective delivery vehicles, entrapment of the miRNA within endocytic compartments, and rapid degradation of miRNA by nucleases. To address these issues, we developed a miRNA delivery strategy that includes (1) a targeting ligand, (2) an endosomal escape agent, nigericin and (3) a chemically modified miRNA. The delivery ligand, DUPA (2-[3-(1,3-dicarboxy propyl) ureido] pentanedioic acid), was selected based on its specificity for prostate-specific membrane antigen (PSMA), a receptor routinely upregulated in prostate cancer-one of the leading causes of cancer death among men. DUPA was conjugated to the tumor suppressive miRNA, miR-34a (DUPA-miR-34a) based on the ability of miR-34a to inhibit prostate cancer cell proliferation. To mediate endosomal escape, nigericin was incorporated into the complex, resulting in DUPA-nigericin-miR-34a. Both DUPA-miR-34a and DUPA-nigericin-miR-34a specifically bound to, and were taken up by, PSMA-expressing cells in vitro and in vivo. And while both DUPA-miR-34a and DUPA-nigericin-miR-34a downregulated miR-34a target genes, only DUPA-nigericin-miR-34a decreased cell proliferation in vitro and delayed tumor growth in vivo. Tumor growth was further reduced using a fully modified version of miR-34a that has significantly increased stability.

Efficient systemic CNS delivery of a therapeutic antisense oligonucleotide with a blood-brain barrier-penetrating ApoE-derived peptide.

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.

Polyphenolic Nanoparticle Platforms (PARCELs) for In Vitro and In Vivo mRNA Delivery.

Despite their successful implementation in the COVID-19 vaccines, lipid nanoparticles (LNPs) still face a central limitation in the delivery of mRNA payloads: endosomal trapping. Improving upon this inefficiency could afford improved drug delivery systems, paving the way toward safer and more effective mRNA-based medicines. Here, we present polyphenolic nanoparticle platforms (PARCELs) as effective mRNA delivery systems. In brief, our investigation begins with a computationally guided structural analysis of 1825 discrete polyphenolic structural data points across 73 diverse small molecule polyphenols and 25 molecular parameters. We then generate structurally diverse PARCELs, evaluating their in vitro mechanism and activity, ultimately highlighting the superior endosomal escape properties of PARCELs relative to analogous LNPs. Finally, we examine the in vivo biodistribution, protein expression, and therapeutic efficacy of PARCELs in mice. In undertaking this approach, the goal of this study is to establish PARCELs as viable delivery platforms for safe and effective mRNA delivery.

Mutational Analysis of RIP Type I Dianthin-30 Suggests a Role for Arg24 in Endocytosis.

Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.

Cetuximab-Toxin Conjugate and NPe6 with Light Enhanced Cytotoxic Effects in Head and Neck Squamous Cell Carcinoma In Vitro.

BACKGROUND: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and irradiation of a specific wavelength to exert cytotoxic effects. To enhance the antitumor effect against head and neck squamous cell carcinoma (HNSCC), we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin (IT) and a PS for PDT and light irradiation. In our prior study, we demonstrated that an immunotoxin (IT) consisting of an anti-ROBO1 antibody conjugated to saporin, when used in combination with the photosensitizer (PS) disulfonated aluminum phthalocyanine (AlPcS2a) and irradiated with light at the appropriate wavelength, resulted in increased cytotoxicity against head and neck squamous cell carcinoma (HNSCC) cells. ROBO1 is a receptor known to be involved in the progression of cancer. In this study, we newly investigate the iTAP targeting epidermal growth factor receptor (EGFR) which is widely used as a therapeutic target for HNSCC. METHODS: We checked the expression of EGFR in HNSCC cell lines, SAS, HO-1-u-1, Sa3, and HSQ-89. We analyzed the cytotoxicity of saporin-conjugated anti-EGFR antibody (cetuximab) (IT-Cmab), mono-L-aspartyl chlorin e6 (NPe6, talaporfin sodium), and light (664 nm) irradiation (i.e., iTAP) in SAS, HO-1-u-1, Sa3, and HSQ-89 cells. RESULTS: EGFR was expressed highly in Sa3, moderately in HO-1-u-1, SAS, and nearly not in HSQ-89. Cmab alone or IT-Cmab alone did not show cytotoxic effects in Sa3, HO-1-u-1, and HSQ-89 cells, which have moderate or low expression levels of EGFR protein. However, the iTAP method enhanced the cytotoxicity of IT-Cmab by the photodynamic effect in Sa3 and HO-1-u-1 cells, which have moderate levels of EGFR expression. CONCLUSION: Our study is the first to report on the iTAP method using IT-Cmab and NPe6 for HNSCC. The cytotoxic effects are enhanced in cell lines with moderate levels of EGFR protein expression, but not in nonexpressing cell lines, which is expected to expand the range of therapeutic windows and potentially reduce complications.

A cleavable peptide adapter augments the activity of targeted toxins in combination with the glycosidic endosomal escape enhancer SO1861.

BACKGROUND: Treatment with tumor-targeted toxins attempts to overcome the disadvantages of conventional cancer therapies by directing a drug's cytotoxic effect specifically towards cancer cells. However, success with targeted toxins has been hampered as the constructs commonly remain bound to the outside of the cell or, after receptor-mediated endocytosis, are either transported back to the cell surface or undergo degradation in lysosomes. Hence, solutions to ensure endosomal escape are an urgent need in treatment with targeted toxins. In this work, a molecular adapter that consists of a cell penetrating peptide and two cleavable peptides was inserted into a targeted toxin between the ribosome-inactivating protein dianthin and the epidermal growth factor. Applying cell viability assays, this study examined whether the addition of the adapter further augments the endosomal escape enhancement of the glycosylated triterpenoid SO1861, which has shown up to more than 1000-fold enhancement in the past. RESULTS: Introducing the peptide adapter into the targeted toxin led to an about 12-fold enhancement in the cytotoxicity on target cells while SO1861 caused a 430-fold increase. However, the combination of adapter and glycosylated triterpenoid resulted in a more than 4300-fold enhancement and in addition to a 51-fold gain in specificity. CONCLUSIONS: Our results demonstrated that the cleavable peptide augments the endosomal escape mediated by glycosylated triterpenoids while maintaining specificity. Thus, the adapter is a promising addition to glycosylated triterpenoids to further increase the efficacy and therapeutic window of targeted toxins.

Overcoming Endosomal Escape Barriers in Gene Drug Delivery Using De Novo Designed pH-Responsive Peptides.

A major challenge in using nanocarriers for intracellular drug delivery is their restricted capacity to escape from endosomes into the cytosol. Here, we significantly enhance the drug delivery efficiency by accurately predicting and regulating the transition pH (pH0) of peptides to modulate their endosomal escape capability. Moreover, by inverting the chirality of the peptide carriers, we could further enhance their ability to deliver nucleic acid drugs as well as antitumor drugs. The resulting peptide carriers exhibit versatility in transfecting various cell types with a high efficiency of up to 90% by using siRNA, pDNA, and mRNA. In vivo antitumor experiments demonstrate a tumor growth inhibition of 83.4% using the peptide. This research offers a potent method for the rapid development of peptide vectors with exceptional transfection efficiencies for diverse pathophysiological indications.

Selective Intracellular Delivery of Antibodies in Cancer Cells with Nanocarriers Sensing Endo/Lysosomal Enzymatic Activity.

The differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape.

Liter-scale manufacturing of shelf-stable plasmid DNA/PEI transfection particles for viral vector production.

The transfection efficiency and stability of the delivery vehicles of plasmid DNA (pDNA) are critical metrics to ensure high-quality and high-yield production of viral vectors. We previously identified that the optimal size of pDNA/poly(ethylenimine) (PEI) transfection particles is 400-500 nm and developed a bottom-up assembly method to construct stable 400-nm pDNA/PEI particles and benchmarked their transfection efficiency in producing lentiviral vectors (LVVs). Here, we report scale-up production protocols for such transfection particles. Using a two-inlet confined impinging jet (CIJ) mixer with a dual syringe pump set-up, we produced a 1-L batch at a flow rate of 100 mL/min, and further scaled up this process with a larger CIJ mixer and a dual peristaltic pump array, allowing for continuous production at a flow rate of 1 L/min without a lot size limit. We demonstrated the scalability of this process with a 5-L lot and validated the quality of these 400-nm transfection particles against the target product profile, including physical properties, shelf and on-bench stability, transfection efficiency, and LVV production yield in both 15-mL bench culture and 2-L bioreactor runs. These results confirm the potential of this particle assembly process as a scalable manufacturing platform for viral vector production.