Intracellular Delivery Research Articles

Catechin-Functionalized Cationic Lipopolymer Based Multicomponent Nanomicelles for Lung-Targeting Delivery.

Catechins from green tea are one of the most effective natural compounds for cancer chemoprevention and have attracted extensive research. Cancer cell-selective apoptosis-inducing properties of catechins depend on efficient intracellular delivery. However, the low bioavailability limits the application of catechins. Herein, a nano-scaled micellar composite composed of catechin-functionalized cationic lipopolymer and serum albumin is constructed. Cationic liposomes tend to accumulate in the pulmonary microvasculature due to electrostatic effects and are able to deliver the micellar system intracellularly, thus improving the bioavailability of catechins. Albumin in the system acts as a biocompatible anti-plasma absorbent, forming complexes with positively charged lipopolymer under electrostatic interactions, contributing to prolonged in vivo retention. The physicochemical properties of the nano-micellar complexes are characterized, and the antitumor properties of catechin-functionalized materials are confirmed by reactive oxygen species (ROS), caspase-3, and cell apoptosis measurements. The role of each functional module, cationic polymeric liposome, and albumin is revealed by cell penetration, in vivo animal assays, etc. This multicomponent micellar nanocomposite has the potential to become an effective vehicle for the treatment of lung diseases such as pneumonia, lung tumors, sepsis-induced lung injury, etc. This study also demonstrates that it is a great strategy to create a delivery system that is both tissue-targeted and biologically active by combining cationic liposomes with the native bioactive compound catechins.

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.

Liquid Nanoparticles for Nanocatalytic Cancer Therapy.

Nanotechnology is revolutionizing cancer therapy, and catalyzes the emerging of ion-involved cancer-therapeutic modality, which unfortunately suffers from undesirable nanocarriers for efficient intracellular ion delivery. To radically extricate from this critical issue, the glutathione (GSH)-responsive organosilica network is employed to lock the liquid drops at the nanoscale via a general bottom-up strategy to achieve the systemic delivery of "ion drugs". In this work, a sulfate radical generation donor (Na2 S2 O8 ), as a paradigm "ion drug", is entrapped into this liquid nanoparticle for efficiently delivering to the tumor region. After further surface engineering with pH-responsive tannic acid-Fe2+ framework, these liquid nanoparticles achieve tumor-microenvironmental pH/GSH-dual responsive ion release (Fe2+ /Na+ /S2 O8 2- ) after reaching the tumor sites, where the Fe2+ further triggers S2 O8 2- to generate toxic •SO4 - and •OH, effectively executing cancer cell ferroptosis (Fe2+ , reactive oxygen species-ROS) and pyroptosis (Na+ , ROS). Such a tumor-responsive/specific liquid nanoplatform is highly instructive for further ion-mediated nanomedicine and disease treatment.

A Bacterial mRNA-Lysis-Mediated Cargo Release Vaccine System for Regulated Cytosolic Surveillance and Optimized Antigen Delivery.

Engineered vector-based in vivo protein delivery platforms have made significant progress for both prophylactic and therapeutic applications. However, the lack of effective release strategies results in foreign cargo being trapped within the vector, restricting the provision of significant performance benefits and enhanced therapeutic results compared to traditional vaccines. Herein, the development of a Salmonella mRNA interferase regulation vector (SIRV) system is reported to overcome this challenge. The genetic circuits are engineered that (1) induce self-lysis to release foreign antigens into target cells and (2) activate the cytosolic surveillance cGAS-STING axis by releasing DNA into the cytoplasm. Delayed synthesis of the MazF interferase regulates differential mRNA cleavage, resulting in a 36-fold increase in the delivery of foreign antigens and modest activation of the inflammasome, which collectively contribute to the marked maturation of antigen-presenting cells (APCs). Bacteria delivering the protective antigen SaoA exhibits excellent immunogenicity and safety in mouse and pig models, significantly improving the survival rate of animals challenged with multiple serotypes of Streptococcus suis. Thus, the SIRV system enables the effective integration of various modular components and antigen cargos, allowing for the generation of an extensive range of intracellular protein delivery systems using multiple bacterial species in a highly efficient manner.

Redox-Responsive Polymeric Nanoparticle for Nucleic Acid Delivery and Cancer Therapy: Progress, Opportunities, and Challenges.

Cancer development and progression of cancer are closely associated with the activation of oncogenes and loss of tumor suppressor genes. Nucleic acid drugs (e.g., siRNA, mRNA, and DNA) are widely used for cancer therapy due to their specific ability to regulate the expression of any cancer-associated genes. However, nucleic acid drugs are negatively charged biomacromolecules that are susceptible to serum nucleases and cannot cross cell membrane. Therefore, specific delivery tools are required to facilitate the intracellular delivery of nucleic acid drugs. In the past few decades, a variety of nanoparticles (NPs) are designed and developed for nucleic acid delivery and cancer therapy. In particular, the polymeric NPs in response to the abnormal redox status in cancer cells have garnered much more attention as their potential in redox-triggered nanostructure dissociation and rapid intracellular release of nucleic acid drugs. In this review, the important genes or signaling pathways regulating the abnormal redox status in cancer cells are briefly introduced and the recent development of redox-responsive NPs for nucleic acid delivery and cancer therapy is systemically summarized. The future development of NPs-mediated nucleic acid delivery and their challenges in clinical translation are also discussed.

Dependence of sonoporation efficiency on microbubble size: An in vitro monodisperse microbubble study

Sonoporation is the process where intracellular drug delivery is facilitated by ultrasound-driven microbubble oscillations. Several mechanisms have been proposed to relate microbubble dynamics to sonoporation including shear and normal stress. The present work aims to gain insight into the role of microbubble size on sonoporation and thereby into the relevant mechanism(s) of sonoporation. To this end, we measured the sonoporation efficiency while varying microbubble size using monodisperse microbubble suspensions. Sonoporation experiments were performed in vitro on cell monolayers using a single ultrasound pulse with a fixed frequency of 1 MHz while the acoustic pressure amplitude and pulse length were varied at 250, 500, and 750 kPa, and 10, 100, and 1000 cycles, respectively. Sonoporation efficiency was quantified using flow cytometry by measuring the FITC-dextran (4 kDa and 2 MDa) fluorescence intensity in 10,000 cells per experiment to average out inherent variations in the bioresponse. Using ultra-high-speed imaging at 10 million frames per second, we demonstrate that the bubble oscillation amplitude is nearly independent of the equilibrium bubble radius at acoustic pressure amplitudes that induce sonoporation (≥ 500 kPa). However, we show that sonoporation efficiency is strongly dependent on the equilibrium bubble size and that under all explored driving conditions most efficiently induced by bubbles with a radius of 4.7 μm. Polydisperse microbubbles with a typical ultrasound contrast agent size distribution perform almost an order of magnitude lower in terms of sonoporation efficiency than the 4.7-μm bubbles. We elucidate that for our system shear stress is highly unlikely the mechanism of action. By contrast, we show that sonoporation efficiency correlates well with an estimate of the bubble-induced normal stress.

Histidine-Rich Protein Accelerates the Biomineralization of Zeolitic Imidazolate Frameworks for In Vivo Protein Delivery.

Biomineralization of metal-organic frameworks (MOFs) provides a powerful approach for intracellular protein delivery, enabling the study of biological function and therapeutic potential of proteins. However, the potency of this approach is largely challenged by the low efficiency of current strategies for interfacing proteins with MOFs for biomineralization and intracellular delivery. Here, we report a versatile and convenient biomineralization strategy for the rapid encapsulation and enhanced delivery of proteins using MOFs, accelerated by histidine-rich proteins. We demonstrate that the histidine-rich green fluorescent protein (H39GFP) can accelerate the biomineralization of MOFs by promoting the coordination between proteins and metal ions, leading to enhanced protein delivery efficiency up to 15-fold. Moreover, we show that the delivery of H39GFP-fused cytotoxic ribonuclease and bacterial-derived RAS protease can effectively inhibit tumor cell growth. The strategy of promoting the biomineralization of MOFs via histidine-rich proteins for enhanced intracellular delivery could be expanded to other biomacromolecules, advancing their therapeutic potential and the biomedical scope of MOFs.

Nanomechanical action opens endo-lysosomal compartments

Endo-lysosomal escape is a highly inefficient process, which is a bottleneck for intracellular delivery of biologics, including proteins and nucleic acids. Herein, we demonstrate the design of a lipid-based nanoscale molecular machine, which achieves efficient cytosolic transport of biologics by destabilizing endo-lysosomal compartments through nanomechanical action upon light irradiation. We fabricate lipid-based nanoscale molecular machines, which are designed to perform mechanical movement by consuming photons, by co-assembling azobenzene lipidoids with helper lipids. We show that lipid-based nanoscale molecular machines adhere onto the endo-lysosomal membrane after entering cells. We demonstrate that continuous rotation-inversion movement of Azo lipidoids triggered by ultraviolet/visible irradiation results in the destabilization of the membranes, thereby transporting cargoes, such as mRNAs and Cre proteins, to the cytoplasm. We find that the efficiency of cytosolic transport is improved about 2.1-fold, compared to conventional intracellular delivery systems. Finally, we show that lipid-based nanoscale molecular machines are competent for cytosolic transport of tumour antigens into dendritic cells, which induce robust antitumour activity in a melanoma mouse model.

The power of sulfhydryl groups: Thiolated lipid-based nanoparticles enhance cellular uptake of nucleic acids

AimThe aim of the study was to evaluate the effect of thiolation of lipid-based nanoparticles (LNPs) on cellular uptake of nucleic acids. MethodsA thiolated surfactant was synthesized by binding palmitic acid covalently to cysteine. Green fluorescent protein (GFP) encoding plasmid DNA (pDNA) was used as model nucleic acid and incorporated via hydrophobic ion-pairing with a cationic cholesterol derivate (DcCholesterol) in LNPs that were prepared by solvent injection method using the thiolated surfactant for surface decoration. LNPs were characterized regarding size, polydispersity index, zeta potential and stability in biorelevant media. The endosomal escape properties of LNPs were evaluated by erythrocytes interaction studies. Cell viability and transfection efficiency on HEK293 cells were investigated. ResultsThe structure of the thiolated surfactant was confirmed by 1H NMR and FT-IR. LNPs containing the nucleic acid-DcCholesterol complex with and without the thiolated surfactant were developed and displayed sizes in the range from 173 nm to 233 nm with a narrow size distribution (PDI < 0.3) and a negative zeta potential. LNPs showed no significant increase in size after 4 h of incubation in artificial body fluids. Erythrocytes interaction studies revealed enhanced endosomal escape properties for thiolated LNPs compared to non-thiolated LNPs. LNPs showed in concentrations lower than 0.74 mg/mL cell viability ≥ 80 %. Transfection studies on HEK cells with thiolated LNPs compared to non-thiolated LNP showed a 4.6-fold higher expression of GFP. ConclusionSurface thiolation of LNPs represents a promising tool for enhancing intracellular nucleic acid delivery of LNPs.

Recent Advances in Mesoporous Silica Nanoparticles Delivering siRNA for Cancer Treatment.

Silencing genes using small interfering (si) RNA is a promising strategy for treating cancer. However, the curative effect of siRNA is severely constrained by low serum stability and cell membrane permeability. Therefore, improving the delivery efficiency of siRNA for cancer treatment is a research hotspot. Recently, mesoporous silica nanoparticles (MSNs) have emerged as bright delivery vehicles for nucleic acid drugs. A comprehensive understanding of the design of MSN-based vectors is crucial for the application of siRNA in cancer therapy. We discuss several surface-functionalized MSNs' advancements as effective siRNA delivery vehicles in this paper. The advantages of using MSNs for siRNA loading regarding considerations of different shapes, various options for surface functionalization, and customizable pore sizes are highlighted. We discuss the recent investigations into strategies that efficiently improve cellular uptake, facilitate endosomal escape, and promote cargo dissociation from the MSNs for enhanced intracellular siRNA delivery. Also, particular attention was paid to the exciting progress made by combining RNAi with other therapies to improve cancer therapeutic outcomes.

Human β-Defensin 23 as a Carrier for In Vitro and In Vivo Delivery of mRNA.

The successful application of mRNA therapeutics hinges on the effective intracellular delivery of mRNA both in vitro and in vivo. However, this remains a formidable challenge due to the polyanionic nature, longitudinal shape, and low nuclease resistance of mRNA. In this study, we introduce a novel mRNA delivery platform utilizing a human β-defensin peptide, hBD23. The positive charge of hBD23 allows it to form nanocomplexes with mRNA, facilitating cellular uptake and providing protection against serum nucleases. When optimized for peptide-to-mRNA (N/P) ratios, these hBD23/mRNA complexes demonstrated efficient cellular delivery and subsequent protein expression both in vitro and in vivo. Importantly, as hBD23 is human derived, the complexes exhibited minimal cytotoxicity and immunogenicity. Given its high biocompatibility and delivery efficiency, hBD23 represents a promising platform for the in vitro and in vivo delivery of mRNA.

Methotrexate-conjugated zinc oxide nanoparticles exert a substantially improved cytotoxic effect on lung cancer cells by inducing apoptosis.

In the current study, we report the synthesis of methotrexate-conjugated zinc oxide nanoparticles (MTX-ZnONPs) and their high efficacy against lung cancer cells. Conjugation of MTX with ZnONPs was authenticated by UV-vis spectroscopy, dynamic light scattering (DLS), Fourier-transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM). This drug-nanoconjugate also showed high drug-loading efficiency. The therapeutic efficacy of MTX-ZnONPs was further tested in vitro against A549 cells, and the results of MTT and LDH release assays showed that MTX-ZnONPs, in addition to free MTX, were efficient in exerting cytotoxic effect on A549 cells; however, the effectiveness of MTX-ZnONPs was found to be considerably enhanced at very low doses compared to that of free MTX. Moreover, ZnONPs alone significantly inhibited the cell viability of A549 cells at a much higher concentration compared to MTX-ZnONPs and MTX. Furthermore, the cytomorphology of A549 cells was characterized by cellular shrinkage and detachment from the surface in all the treatment groups. Similarly, A549 cells, in all the treatment groups, showed fragmented and condensed nuclei, indicating the initiation of apoptosis. Mitochondrial membrane potential (ψm) in A549 cells showed a gradual loss in all the treatment groups. Results of the qualitative and quantitative analyses depicted increased reactive oxygen species (ROS) levels in A549 cells. The results of the caspase activity assay showed that MTX-ZnONPs andfree MTX caused significant activation of caspase-9, -8, and -3 in A549 cells; however, the effect of MTX-ZnONPs was more profound at very low doses compared to that of free MTX. Thus, our results showed high efficacy of MTX-ZnONPs, suggesting efficient intracellular delivery of the drug by ZnONPs as nanocarriers.

Bio-orthogonal engineered peptide: A multi-functional strategy for the gene therapy of osteoporotic bone loss

Osteoporosis is a degenerative disease affecting millions of elderly people globally and increases the risk of bone fractures due to the reduced bone density. Drugs are normally prescribed to treat osteoporosis, especially after surgical treatment of osteoporotic fractures. However, many anti-osteoporotic drugs produce deleterious side effects. The recent development of gene therapy utilizing oligonucleotides (ONs) has spurred the development of new therapies for osteoporosis. Nevertheless, most ONs lack the capability of cell penetration and lysosome escape and hence, intracellular delivery of ON remains a challenge. Herein, a novel strategy is demonstrated to efficiently deliver ON to cells by combining ON with the cell-penetrating peptide (CPP) via the bio-orthogonal click reaction. Several dopamine (DOPA) groups are also introduced into the fabricated peptide to scavenge intracellular reactive oxygen species (ROS). Owing to favorable properties such as good cytocompatibility, cell penetration, lysosome escape, ROS scavenging, and osteoclastogenesis suppression, the hybrid CPP-DOPA-ON peptide improves the osteoporotic conditions significantly in vivo even when bone implants are involved. This strategy has great potential in the treatment of osteoporosis and potentially broadens the scope of gene therapy.

Topoisomeric Membrane-Active Peptides: A Review of the Last Two Decades.

In recent decades, bioactive peptides have been gaining recognition in various biomedical areas, such as intracellular drug delivery (cell-penetrating peptides, CPPs) or anti-infective action (antimicrobial peptides, AMPs), closely associated to their distinct mode of interaction with biological membranes. Exploiting the interaction of membrane-active peptides with diverse targets (healthy, tumoral, bacterial or parasitic cell membranes) is opening encouraging prospects for peptides in therapeutics. However, ordinary peptides formed by L-amino acids are easily decomposed by proteases in biological fluids. One way to sidestep this limitation is to use topoisomers, namely versions of the peptide made up of D-amino acids in either canonic (enantio) or inverted (retroenantio) sequence. Rearranging peptide sequences in this fashion provides a certain degree of native structure mimicry that, in appropriate contexts, may deliver desirable biological activity while avoiding protease degradation. In this review, we will focus on recent accounts of membrane-active topoisomeric peptides with therapeutic applications as CPP drug delivery vectors, or as antimicrobial and anticancer candidates. We will also discuss the most common modes of interaction of these peptides with their membrane targets.

Diethylaminoethyl-dextran and monocyte cell membrane coated 1,8-cineole delivery system for intracellular delivery and synergistic treatment of atherosclerosis

We have developed a biomimetic delivery system termed the Monocyte Cell Membrane-Coated 1,8-Cineole Biomimetic Delivery System (MM-CIN-BDS or BDS), which integrates diethylaminoethyl-dextran (DEAE) and monocyte cell membrane (MM). This innovative approach enhances the cellular uptake efficiency of 1,8-cineole (CIN) and facilitates targeted therapy for atherosclerosis. Our findings demonstrate the successful modification of the drug carrier with DEAE and MM, as validated by measurements of particle size, zeta potential, microscopic morphology, and western blotting analyses. Notably, cellular uptake experiments unveil a significant enhancement in cellular uptake efficiency due to DEAE modification. However, the introduction of monocyte cell membranes diminishes this effect in normal human umbilical vein endothelial cells (HUVECs), although this efficiency is notably restored in HUVECs activated with lipopolysaccharide (LPS). Through in vivo imaging investigations, we observe that the MM coating augments distribution in the spleen, brain, and atherosclerotic plaques, while concurrently diminishing distribution in the heart and kidneys. Animal studies corroborate these findings, illustrating that MM-CIN-BDS treatment curtails lipid parameters, dampens the expression of inflammatory factors and proteins, mitigates vascular tissue damage, and ultimately reduces the extent of atherosclerotic lesion areas. To encapsulate, DEAE emerges as an especially adept agent for modifying drug carriers with suboptimal cellular uptake efficiency in the realm of cardiovascular diseases. The potential therapeutic promise of MM-CIN-BDS for atherosclerosis treatment is evident from our research.

Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity.

Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.

Immobilization of horseradish peroxidase on metal-organic framework to improve enzyme activity for enhanced chemodynamic therapy

Bio-enzymes have the advantages of strong substrate specificity, high catalytic efficiency, and minimal toxic side effects, making them promising drugs in cancer therapy. However, the poor stability and cellular penetrability of uncoated protein in the physiological environment severely restricts the direct application of Bio-enzyme. To address it, we report a metal-organic framework (MOF), Hf-DBA (H2DBA, biphenyl carboxylic acid ligands). The morphology of the Hf-DBA was revealed by TEM and the diameter was in the range of 200 to 350 nm. Hf-DBA acted a carrier for intracellular delivery and protection of horseradish peroxidase (HRP). The prepared HRP@Hf-DBA can catalyze the excess H2O2 in the tumor cells to generation of •OH for chemodynamic therapy (CDT). Compared with free HRP, the catalytic activity of HRP@Hf-DBA is significantly improved, and the optimal catalytic conditions are explored. The catalytic stability of HRP@Hf-DBA remained above 70% after 12 cycles of catalysis. After treatment with HRP@Hf-DBA, the apoptosis rates of A549 and Hela cells was 71.64%, and 76.86%. The results in vitro show that HRP@Hf-DBA can effectively inhibit the growth of tumor cells through enhanced CDT.

Inkjet-Based Intracellular Delivery System that Effectively Utilizes Cell-Penetrating Peptides for Cytosolic Introduction of Biomacromolecules through the Cell Membrane.

In the drug delivery system, the cytosolic delivery of biofunctional molecules such as enzymes and genes must achieve sophisticated activities in cells, and microinjection and electroporation systems are typically used as experimental techniques. These methods are highly reliable, and they have high intracellular transduction efficacy. However, a high degree of proficiency is necessary, and induced cytotoxicity is considered as a technical problem. In this research, a new intracellular introduction technology was developed through the cell membrane using an inkjet device and cell-penetrating peptides (CPPs). Using the inkjet system, the droplet volume, droplet velocity, and dropping position can be accurately controlled, and minute samples (up to 30 pL/shot) can be carried out by direct administration. In addition, CPPs, which have excellent cell membrane penetration functions, can deliver high-molecular-weight drugs and nanoparticles that are difficult to penetrate through the cell membrane. By using the inkjet system, the CPPs with biofunctional cargo, including peptides, proteins such as antibodies, and exosomes, could be accurately delivered to cells, and efficient cytosolic transduction was confirmed.

Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo.

Inflammation plays a crucial role in the development and progression of many diseases, and is often caused by dysregulation of signalling from pattern recognition receptors, such as TLRs. Inhibition of key protein-protein interactions is an attractive target for treating inflammation. Recently, we demonstrated that the signalling lymphocyte activation molecule family 1 (SLAMF1) positively regulates signalling downstream of TLR4 and identified the interaction interface between SLAMF1 and the TLR4 adaptor protein TRIF-related adapter molecule (TRAM). Based on these findings, we developed a SLAMF1-derived peptide, P7, which is linked to a cell-penetrating peptide for intracellular delivery. We found that P7 peptide inhibits the expression and secretion of IFNβ and pro-inflammatory cytokines (TNF, IL-1β, IL-6) induced by TLR4, and prevents death in mice subjected to LPS shock. The mechanism of action of P7 peptide is based on interference with several intracellular protein-protein interactions, including TRAM-SLAMF1, TRAM-Rab11FIP2, and TIRAP-MyD88 interactions. Overall, P7 peptide has a unique mode of action and demonstrates high efficacy in inhibiting TLR4-mediated signalling in vitro and in vivo.

Enhancing Antisense Oligonucleotide-Based Therapeutic Delivery with DG9, a Versatile Cell-Penetrating Peptide.

Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising biological and pharmacological properties for antisense applications. Despite their great potential, the efficient delivery of these therapeutic agents to target cells remains a major obstacle to their widespread use. Cellular uptake of naked PMO is poor. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular uptake and intracellular delivery of oligonucleotide-based drugs. Among these, the DG9 peptide has been identified as a versatile CPP with remarkable potential for enhancing the delivery of ASO-based therapeutics due to its unique structural features. Notably, in the context of phosphorodiamidate morpholino oligomers (PMOs), DG9 has shown promise in enhancing delivery while maintaining a favorable toxicity profile. A few studies have highlighted the potential of DG9-conjugated PMOs in DMD (Duchenne Muscular Dystrophy) and SMA (Spinal Muscular Atrophy), displaying significant exon skipping/inclusion and functional improvements in animal models. The article provides an overview of a detailed understanding of the challenges that ASOs face prior to reaching their targets and continued advances in methods to improve their delivery to target sites and cellular uptake, focusing on DG9, which aims to harness ASOs' full potential in precision medicine.