Endosomal Escape Research Articles

Brain targeted biomimetic siRNA nanoparticles for drug resistance glioblastoma treatment

Glioblastoma multiforme (GBM), the most aggressive intracranial neoplasm, remains incurable at present, primarily due to drug resistance, which significantly contributes to elevated recurrence rates and dismal prognosis. Signal transducer and activator of transcription 3 (STAT3) is a critical gene closely associated with GBM drug resistance and the progression of GBM stem cells (GSCs), making it a promising therapeutic target. In this study, we developed cancer cell membrane-cloaked biomimetic nanoparticles to deliver STAT3 siRNA to reverse drug resistance in homologous GBM. These biomimetic nanoparticles leverage homotypic targeting, rapid endosome escape, and fast siRNA release, leading to efficient in vitro STAT3 knockdown in both temozolomide-resistant U251-TR cells and X01 GSCs. Moreover, benefited from the membrane functionalization, significant prolonged blood circulation, improved blood brain barrier (BBB) penetration and GBM tumor accumulation are achieved by these siRNA biomimetic nanoparticles. Importantly, these nanoparticles effectively inhibit tumor proliferation, significantly extending median survival time in orthotopic U251-TR (43.5 d versus 20 d for PBS control) and X01 GSC-bearing mouse xenografts (52 d versus 19.5 d for PBS control). Altogether, this biomimetic siRNA platform offers a promising strategy for gene therapy targeting drug-resistant GBM.

Tackling Anticancer Drug Resistance and Endosomal Escape in Aggressive Brain Tumors Using Bioelectronics.

Resistance mechanisms in brain tumors, such as medulloblastoma and glioblastoma, frequently involve the entrapment of chemotherapeutic agents within endosomes and the extracellular expulsion of drugs. These barriers to effective treatment are exacerbated in nanotechnology-based drug delivery systems, where therapeutic nanoparticles often remain confined within endosomes, thus diminishing their therapeutic efficacy. Addressing this challenge necessitates the development of novel strategies to enhance the efficiency of cancer therapies. This study tests the hypothesis that external electrical stimuli can modulate intracellular trafficking of chemotherapeutic drugs in common malignant brain tumors in children (medulloblastoma) and adults (glioblastoma) by using gold nanoparticles (GNPs). In our experiments, alternating current (AC) stimulation ranging from 1 kHz to 5 MHz and at a strength of 1 V/cm significantly reduced cell viability in drug-resistant medulloblastoma and enhanced delivery of GNPs in glioblastoma. Low-frequency AC resulted in a 50% increase in apoptosis compared to controls and an 8-fold increase in cell death in cisplatin-resistant medulloblastoma cells, accompanied by a substantial reduction in EC50 from 2.5 to 0.3 μM. Similarly, vincristine-resistant cells demonstrated a 4-fold enhancement in drug sensitivity. Furthermore, high-frequency AC facilitated a significant increase from 20 to 75% in the endosomal escape of GNPs in glioblastoma cells. These findings underscore the potential of AC to selectively disrupt cancer cell resistance mechanisms and bolster the efficacy of nanoparticle-based therapies. The results indicate the effectiveness of AC stimulation in circumventing the limitations inherent in current nanotechnology-based drug delivery systems but also illustrates its transformative potential for treating aggressive, drug-resistant brain tumors.

Elucidating siRNA Cellular Delivery Mechanism Mediated by Quaternized Starch Nanoparticles.

Starch-based nanoparticles are highly utilized in the realm of drug delivery taking advantage of their biocompatibility and biodegradability. Studies have utilized Quaternized starch (Q-starch) for small interfering RNA (siRNA) delivery, in which quaternary amines enable interaction with negatively charged siRNA, resulting in self-assembly complexation. Although reports present numerous applications, the demonstrated efficacy is nonetheless limited due to undiscovered cellular mechanistic delivery. In this study, a deep dive into Q-starch/siRNA complexes' cellular mechanism and kinetics at the cellular level is revealed using single-particle tracking and cell population level using imaging flow cytometry. Uptake studies depict the efficient cellular internalization via endocytosis while a significant fraction of complexes' intracellular fate is lysosome. Utilizing single-particle tracking, it is found that an average of 15% of cellular detected complexes escape the endosome which holds the potential for the integration in the cytoplasmatic gene silencing mechanism. Additional experimental manipulations (overcoming endosomal escape) demonstrate that the complex's disassembly is the rate-limiting step, correlating Q-starch's structure-function properties as siRNA carrier. Structure-function properties accentuating the high affinity of the interaction between Q-starch's quaternary groups and siRNA's phosphate groups that results in low release efficiency. However, low-frequency ultrasound (20kHz) application may have induced siRNA release resulting in faster gene silencing kinetics.

Delivery of Avocado Seed Extract Using Novel Charge-Switchable Mesoporous Silica Nanoparticles with Galactose Surface Modified to Target Sorafenib-Resistant Hepatocellular Carcinoma.

Sorafenib-resistant (SR) hepatocellular carcinoma (HCC) is a current serious problem in liver cancer treatment. Numerous phytochemicals derived from plants exhibit anticancer activity but have never been tested against drug-resistant cells. Avocado seed extract (APE) isolated by maceration was analysed for its phytochemical composition and anticancer activity. Novel design charge-switchable pH-responsive nanocarriers of aminated mesoporous silica nanoparticles with conjugated galactose (GMSN) were synthesised for delivering APE and their physicochemical properties were characterized. The drug loading efficiency (%LE) and entrapment efficiency (%EE) were evaluated. Anticancer activity of APE loaded GMSN was measured against HCC (HepG2, Huh-7) and SR-HCC (SR-HepG2). Anticancer activity of APE against non-resistant HepG2 (IC50 50.9 ± 0.83 μg mL-1), Huh-7 (IC50 42.41 ± 1.88 μg mL-1), and SR-HepG2 (IC50 62.58 ± 2.29 μg mL-1) cells was confirmed. The APE loaded GMSN had a diameter of 131.41 ± 14.41 nm with 41.08 ± 2.09%LE and 44.96 ± 2.26%EE. Galactose functionalization (55%) did not perturb the original mesoporous structure. The GMSN imparted positive surface charges, 10.3 ± 0.61mV at acidic medium pH 5.5 along with rapid release of APE 45% in 2h. The GMSN boosted cellular uptake by HepG2 and SR-HepG2 cells, whereas the amine functionalized facilitated their endosomal escape. Their anticancer activity was demonstrated in non-resistant HCC and SR-HCC cells with IC50 values at 30.73 ± 3.14 (HepG2), 21.86 ± 0.83 (Huh-7), 35.64 ± 1.34 (SR-HepG2) μg mL-1, respectively, in comparison to the control and non-encapsulated APE. APE loaded GMSN is highly effective against both non-resistant HCC and SR-HCC and warrants further in vivo investigation.

Combinatorial design of siloxane-incorporated lipid nanoparticles augments intracellular processing for tissue-specific mRNA therapeutic delivery.

Systemic delivery of messenger RNA (mRNA) for tissue-specific targeting using lipid nanoparticles (LNPs) holds great therapeutic potential. Nevertheless, how the structural characteristics of ionizable lipids (lipidoids) impact their capability to target cells and organs remains unclear. Here we engineered a class of siloxane-based ionizable lipids with varying structures and formulated siloxane-incorporated LNPs (SiLNPs) to control in vivo mRNA delivery to the liver, lung and spleen in mice. The siloxane moieties enhance cellular internalization of mRNA-LNPs and improve their endosomal escape capacity, augmenting their mRNA delivery efficacy. Using organ-specific SiLNPs to deliver gene editing machinery, we achieve robust gene knockout in the liver of wild-type mice and in the lungs of both transgenic GFP and Lewis lung carcinoma (LLC) tumour-bearing mice. Moreover, we showed effective recovery from viral infection-induced lung damage by delivering angiogenic factors with lung-targeted Si5-N14 LNPs. We envision that our SiLNPs will aid in the clinical translation of mRNA therapeutics for next-generation tissue-specific protein replacement therapies, regenerative medicine and gene editing.

Optimized lipid nanoparticles enable effective CRISPR/Cas9-mediated gene editing in dendritic cells for enhanced immunotherapy

Immunotherapy has emerged as a revolutionary approach to treat immune-related diseases. Dendritic cells (DCs) play a pivotal role in orchestrating immune responses, making them an attractive target for immunotherapeutic interventions. Modulation of gene expression in DCs using genome editing techniques, such as the CRISPR-Cas system, is important for regulating DC functions. However, the precise delivery of CRISPR-based therapies to DCs has posed a significant challenge. While lipid nanoparticles (LNPs) have been extensively studied for gene editing in tumor cells, their potential application in DCs has remained relatively unexplored. This study investigates the important role of cholesterol in regulating the efficiency of BAMEA-O16B lipid-assisted nanoparticles (BLANs) as carriers of CRISPR/Cas9 for gene editing in DCs. Remarkably, BLANs with low cholesterol density exhibit exceptional mRNA uptake, improved endosomal escape, and efficient single-guide RNA release capabilities. Administration of BLANmCas9/gPD-L1 results in substantial PD-L1 gene knockout in conventional dendritic cells (cDCs), accompanied by heightened cDC1 activation, T cell stimulation, and significant suppression of tumor growth. The study underscores the pivotal role of cholesterol density within LNPs, revealing potent influence on gene editing efficacy within DCs. This strategy holds immense promise for the field of cancer immunotherapy, offering a novel avenue for treating immune-related diseases.

MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo.

AbstractThere is enormous interest in strategies to efficiently traffic biologics–proteins, nucleic acids, and complexes thereof–into the mammalian cell cytosol and internal organelles. Not only must these materials reach the appropriate cellular locale fully intact and in therapeutically relevant concentrations, they must also retain activity upon arrival. The question of residual activity is especially critical when delivery involves exposure to the late endocytic pathway, whose acidic lumenal environment can denature and/or degrade internalized material. ZF5.3 is a compact, stable, rationally designed mini-protein that efficiently escapes intact from late endocytic vesicles, with or without covalently linked protein cargo. Here, using insights from mechanistic studies on the pathway of endosomal escape and classic knowledge regarding the bioinorganic chemistry of zinc(II) coordination in small proteins, we re-designed the sequence of ZF5.3 to successfully alter the timing (but not the efficiency) of endosomal escape. The new mini-protein we describe, AV5.3, escapes earlier than ZF5.3 along the endocytic pathway with no loss in efficiency, with or without enzyme cargo. More importantly, earlier endosomal escape translates into higher enzymatic activity upon arrival in the cytosol. Delivery of the pH-sensitive protein dihydrofolate reductase (DHFR) with AV5.3 results in substantial catalytic activity in the cytosol, whereas delivery with ZF5.3 does not. The activity of AV5.3-DHFR upon delivery is sufficient to rescue a genetic DHFR deletion in CHO cells. This work provides evidence that programmed trafficking through the endosomal pathway is a viable strategy for the efficient cytosolic delivery of therapeutic proteins.Abstract Figure

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.

Enhancing RNA-lipid nanoparticle delivery: Organ- and cell-specificity and barcoding strategies

Recent advancements in RNA therapeutics highlight the critical need for precision gene delivery systems that target specific organs and cells. Lipid nanoparticles (LNPs) have emerged as key vectors in delivering mRNA and siRNA, offering protection against enzymatic degradation, enabling targeted delivery and cellular uptake, and facilitating RNA cargo release into the cytosol. This review discusses the development and optimization of organ- and cell-specific LNPs, focusing on their design, mechanisms of action, and therapeutic applications. We explore innovations such as DNA/RNA barcoding, which facilitates high-throughput screening and precise adjustments in formulations. We address major challenges, including improving endosomal escape, minimizing off-target effects, and enhancing delivery efficiencies. Notable clinical trials and recent FDA approvals illustrate the practical applications and future potential of LNP-based RNA therapies. Our findings suggest that while considerable progress has been made, continued research is essential to resolve existing limitations and bridge the gap between preclinical and clinical evaluation of the safety and efficacy of RNA therapeutics. This review highlights the dynamic progress in LNP research. It outlines a roadmap for future advancements in RNA-based precision medicine.

Cell-Penetrating Peptides Translocate across the Plasma Membrane by Inducing Vesicle Budding and Collapse.

Cell-penetrating peptides (CPPs) enter the cell by two different mechanisms-endocytosis followed by endosomal escape and direct translocation at the plasma membrane. The mechanism of direct translocation remains unresolved. In this work, the direct translocation of nonaarginine (R9) and two cyclic CPPs (CPP12 and CPP17) into Jurkat cells was monitored by time-lapse confocal microscopy. Our results provide direct evidence that all three CPPs translocate across the plasma membrane by a recently discovered vesicle budding-and-collapse (VBC) mechanism. Membrane translocation is preceded by the formation of nucleation zones. Up to four different types of nucleation zones and three variations of the VBC mechanism were observed. The VBC mechanism reconciles the enigmatic and conflicting observations in the literature.

Novel Endogenous Engineering Platform for Robust Loading and Delivery of Functional mRNA by Extracellular Vesicles.

Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a plethora of clinical applications. For in vivo functionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, a bioengineering platform for efficient mRNA loading and functional delivery using bionormal nanoparticles, extracellular vesicles (EVs), is established by expressing a highly specific RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA. The additional combination with a fusogenic endosomal escape moiety, Vesicular Stomatitis Virus Glycoprotein, enables functional mRNA delivery in vivo at doses substantially lower than currently used clinically with synthetic lipid-based nanoparticles. Importantly, the application of EVs loaded with effective cancer immunotherapy proves highly effective in an aggressive melanoma mouse model. This technology addresses substantial drawbacks currently associated with EV-based nucleic acid delivery systems and is a leap forward to clinical EV applications.

EXTL3 and NPC1 are mammalian host factors for Autographa californica multiple nucleopolyhedrovirus infection

Baculovirus is an obligate parasitic virus of the phylum Arthropoda. Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1−/+ mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.

Synthesis of pH-sensitive polymeric micelle drug carries for potential cancer chemotherapy applications

Alpha mangostin, a natural xanthone derivative from mangosteen fruit pericarp, exhibits antiproliferative and apoptotic effects on different cancer cells by inhibiting cell cycle progression, inducing apoptosis, and modulating signaling pathways. However, this candidate is limited in medical treatment due to the poor water solubility. Nanoparticles have emerged as promising drug delivery systems to improve drug delivery efficiency but one of the problem of nanoparticles is the capability of endosomal escape. Therefore, pH-sensitive polymer nanosystems were utilized for targeted drug delivery, and enhanced efficacy by offering controlled release and endosomal escape capabilities. This study aimed to synthesize PEG- P(Asp-Hyd-Man) copolymer and create micelle for alpha mangostin delivery. The micelles were measured with particle size average of 80.2 ± 15 nm, and the PDI of 0.17. Additionally, the release behavior of alpha mangostin was examined in vitro that show pH-sensitive polymeric micelles rapidly release at pH 5.0 compared with the arterial pH 7.4. The findings of this study are significant in the development of an effective drug delivery system using alpha mangostin and other therapeutic agents.

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.

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.

A Fluorogenic Pseudoinfection Assay to Probe Transfer and Distribution of Influenza Viral Contents to Target Vesicles.

Fusion of enveloped viruses with endosomal membranes and subsequent release of the viral genome into the cytoplasm are crucial to the viral infection cycle. It is often modeled by performing fusion between virus particles and target lipid vesicles. We utilized fluorescence microscopy to characterize the kinetic aspects of the transfer of influenza viral ribonucleoprotein (vRNP) complexes to target vesicles and their spatial distribution within the fused volumes to gain deeper insight into the mechanistic aspects of endosomal escape. The fluorogenic RNA-binding dye QuantiFluor (Promega) was found to be well-suited for direct and sensitive microscopic observation of vRNPs which facilitated background-free detection and kinetic analysis of fusion events on a single particle level. To determine the extent to which the viral contents are transferred to the target vesicles through the fusion pore, we carried out virus-vesicle fusion in a side-by-side fashion. Measurement of the Euclidean distances between the centroids of superlocalized membrane and content dye signals within the fused volumes allowed determination of any symmetry (or the lack thereof) between them as expected in the event of transfer (or the lack thereof) of vRNPs, respectively. We found that, in the case of fusion between viruses and 100 nm target vesicles, ∼39% of the events led to transfer of viral contents to the target vesicles. This methodology provides a rapid, generic, and cell-free way to assess the inhibitory effects of antiviral drugs and therapeutics on the endosomal escape behavior of enveloped viruses.

Determining the Role of Surfactant on the Cytosolic Delivery of DNA Cross-Linked Micelles.

Nucleic Acid Nanocapsules (NANs) are nucleic acid nanostructures that radially display oligonucleotides on the surface of cross-linked surfactant micelles. Their chemical makeup affords the stimuli-responsive release of therapeutically active DNA-surfactant conjugates into the cells. While NANs have so far demonstrated the effective cytosolic delivery of their nucleic acid cargo, as seen indirectly by their gene regulation capabilities, there remain gaps in the molecular understanding of how this process happens. Herein, we examine the enzymatic degradation of NANs and confirm the identity of the DNA-surfactant conjugates formed by using mass spectrometry (MS). With surface enhanced (resonance) Raman spectroscopy (SE(R)RS), we also provide evidence that the energy-independent translocation of such DNA-surfactant conjugates is contingent upon their release from the NAN structure, which, when intact, otherwise buries the hydrophobic surfactant tail in its interior. Such information suggests a critical role of the surfactant in the lipid disruption capability of the DNA surfactant conjugates generated from degradation of the NANs. Using NANs made with different tail lengths (C12 and C10), we show that this mechanism likely holds true despite significant differences in the physical properties (i.e., critical micelle concentration (CMC), surfactants per micelle, Nagg) of the resultant particles (C12 and C10 NANs). While the total cellular uptake efficiencies of C12 and C10 NANs are similar, there were differences observed in their cellular distribution and localized trafficking, even after ensuring that the total concentration of DNA was the same for both particles. Ultimately, C10 NANs appeared less diffuse within cells and colocalized less with lysosomes over time, achieving more significant knockdown of the target gene investigated, suggesting more effective endosomal escape. These differences indicate that the surfactant assembly and disassembly properties, including the number of surfactants per particle and the CMC can have important implications for the cellular delivery efficacy of DNA micelles and surfactant conjugates.

Time-Resolved Inspection of Ionizable Lipid-Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early Endosomal Membrane Mimic.

Advances in lipid nanoparticle (LNP) design have contributed notably to the emergence of the current clinically approved mRNA-based vaccines and are of high relevance for delivering mRNA to combat diseases where therapeutic alternatives are sparse. LNP-assisted mRNA delivery utilizes ionizable lipid-mediated cargo translocation across the endosomal membrane driven by the acidification of the endosomal environment. However, this process occurs at a low efficiency, a few percent at the best. Utilizing surface-sensitive fluorescence microscopy with a single LNP and mRNA resolution, we have investigated pH-controlled interactions between individual LNPs and a planar anionic supported lipid bilayer (SLB) formed on nanoporous silica, mimicking the electrostatic conditions of the early endosomal membrane. For LNPs with an average diameter of 140 nm, fusion with the anionic SLB preferentially occurred when the pH was reduced from 6.6 to 6.0. Furthermore, there was a delay in the onset of LNP fusion after the pH drop, and upon fusion, a significant fraction (>70%) of mRNA was released into the acidic solution representing the endosomal lumen, while a fraction of mRNA remained bound to the SLB even after reversing the pH to neutral cytosolic conditions. Finally, a comparison of the fusion efficiency of two LNP formulations with different surface concentrations of gel-forming lipids correlated with differences in the protein translation efficiency previously observed in human primary cell transfection studies. Together, these findings emphasize the relevance of biophysical investigations of ionizable lipid-containing LNP-assisted mRNA delivery mechanisms while potentially also offering means to optimize the design of LNPs with enhanced endosomal escape capabilities.

Assessment of CRISPRa-mediated gdnf overexpression in an In vitro Parkinson's disease model.

Parkinson's disease (PD) presents a significant challenge in medical science, as current treatments are limited to symptom management and often carry significant side effects. Our study introduces an innovative approach to evaluate the effects of gdnf overexpression mediated by CRISPRa in an in vitro model of Parkinson's disease. The expression of gdnf can have neuroprotective effects, being related to the modulation of neuroinflammation and pathways associated with cell survival, differentiation, and growth. We have developed a targeted delivery system using a magnetite nanostructured vehicle for the efficient transport of genetic material. This system has resulted in a substantial increase, up to 200-fold) in gdnf expression in an In vitro model of Parkinson's disease using a mixed primary culture of astrocytes, neurons, and microglia. The delivery system exhibits significant endosomal escape of more than 56%, crucial for the effective delivery and activation of the genetic material within cells. The increased gdnf expression correlates with a notable reduction in MAO-B complex activity, reaching basal values of 14.8μU/μg of protein, and a reduction in reactive oxygen species. Additionally, there is up to a 34.6% increase in cell viability in an In vitro Parkinson's disease model treated with the neurotoxin MPTP. Our study shows that increasing gdnf expression can remediate some of the cellular symptoms associated with Parkinson's disease in an in vitro model of the disease using a novel nanostructured delivery system.