Figure 4
Enhanced cellular retention and efficacy of siAF647-LNPs in NPC1-deficient MEFs. (a) Immunoblot analysis with anti-NPC1 antibody was used to validate wild-type and NPC1-deficient MEFs. (b) Automated confocal microscopy on NPC1+/+ or NPC1−/− MEFs exposed to different concentrations of labeled LNPs imaged and quantified 24 h after incubation. Inset from a representative image (100 nM) shows siRNA accumulation in individual cells. (c) Flow cytometry analysis on LNP-siRNA uptake in (i) NPC1+/+ or NPC1−/− cells or in (ii) NPC1−/− cells transfected with pEGFP or pNPC1-GFP. The mean fluorescent intensity represents LNP uptake. (d) NPC1−/− cells treated with LNP-AF647-siRNA (red) (3-h pulse, 30-min chase) and immunostained with anti-LBPA antibody (green). (e) LNPs containing siRNA against β integrin were added to wild-type or NPC1-deficient MEFs as in b, mRNA levels were quantified at 24 h after incubation using branched DNA analysis. The experiment was done in triplicate and the errors are reported as s.e.m. (f) A schematic representation of LNP trafficking (i) in NPC1+/+ and (ii) NPC1−/− cells. Intact cationic LNPs enter through macropinocytosis (1); a small fraction of LNPs transport from macropinosomes to the endocytic recycling compartment (ERC) (2) whereas the majority is directed to late endosomes (3). Late endosome sort LNPs to lysosomes for degradation or utilize multiple recycling pathways to traffic them to the extracellular milieu. These mechanisms include recycling through transport to the ER-Golgi route (4) or direct fusion of late endosomes containing multivesicular bodies, with the plasma membrane (Exosomes secretion) (5). In NPC1-deficient cells, the late endosome recycling mechanisms are impaired, causing LNP-siRNA to accumulate in enlarged late endosomes leading to persistent escape of siRNA that improves gene silencing. (Intact nanoparticle-, siRNA complex-). All experiments were performed in triplicate. Error bars, mean ± s.e.m.
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Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated h...
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... another catabolic process that routes cytosolic proteins to the lysosomes 23,24 , was apparently not involved in LNP-mediated gene silencing as LNPs showed little co-localization with markers of autophagy ( Supplementary Fig. 4a,b). Autophagy-deficient cells (Atg5 −/− ) 24 showed little if any differences in LNP-mediated gene silencing when compared to autophagy competent (Atg5 +/+ ) cells (Supplementary Fig. 4c). ...
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... another catabolic process that routes cytosolic proteins to the lysosomes 23,24 , was apparently not involved in LNP-mediated gene silencing as LNPs showed little co-localization with markers of autophagy ( Supplementary Fig. 4a,b). Autophagy-deficient cells (Atg5 −/− ) 24 showed little if any differences in LNP-mediated gene silencing when compared to autophagy competent (Atg5 +/+ ) cells (Supplementary Fig. 4c). ...
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... liver and neural degeneration in human patients and animal models [27][28][29] . As the components of an LNP show some similarities to the endog- enous lipids that use NPC1 as a receptor for recycling, we compared LNP retention in mouse embryonic fibroblasts (MEFs) devoid of NPC1 (NPC1 −/− ) with their wild-type counterparts (NPC1 +/+ MEFs) (Fig. 4a). NPC1-deficient cells accumulated cholesterol due to defects in recycling (Supplementary Fig. 6). A markedly increased level (~15-fold) of AF647-siRNA was observed in perinuclear enlarged endosomes in NPC1 −/− compared to NPC1 +/+ MEFs at 24 h after incubation, presumably due to enhanced cellular retention at a wide range of LNP ...
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... NPC1-deficient cells accumulated cholesterol due to defects in recycling (Supplementary Fig. 6). A markedly increased level (~15-fold) of AF647-siRNA was observed in perinuclear enlarged endosomes in NPC1 −/− compared to NPC1 +/+ MEFs at 24 h after incubation, presumably due to enhanced cellular retention at a wide range of LNP concentrations (Fig. 4a,b). Furthermore, enhanced cellular retention was observed in multiple cell types deficient in NPC1, including those isolated from human patients ( Supplementary Fig. 7a,b). We further tested for differences in the kinetics of LNP ; a small fraction of LNPs transport from macropinosomes to the endocytic recycling compartment (ERC) (2) ...
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... cells. NPC1 −/− cells had accumulated more siRNA after 2 h of incubation as compared to NPC1 +/+ cells where at early time points the amount of internalization was not affected. Moreover, a rescue experiment in which we transfected an NPC1-GFP plasmid into NPC1 −/− cells reduced the amount of intracellular LNP-siRNA to that of wild-type cells (Fig. 4c, i,ii). We conclude that the greater amount of siRNA in late endosomes is based on accumulation of LNPs due to defects in constitutively active NPC1-mediated recycling. Small molecules that impair cholesterol metabolism 28 and increase cholesterol accumulation in cells failed to yield a significant increase in LNP accumulation (Supplementary ...
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... interaction with putative intralu- minal domains of NPC1 required for egress of LNPs. NPC1 −/− cells have been reported to have high numbers of enlarged late endosomes, containing intraluminal vesicles enriched in lysobisphosphatidic acid (LBPA) 29 . LNPs accumulated in these structures and co-localized with antibody against LBPA in these cells (Fig. ...
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... hypothesized that enhanced cellular retention could translate to improved gene silencing. LNPs containing β-integrin siRNA were added to either NPC1 +/+ or NPC1 −/− MEFs at different concentra- tions. A substantial improvement in LNP potency was observed in NPC1 −/− cells in comparison to wild-type cells (Fig. 4e). The median inhibitory concentration (IC 50 ) of 1.5 nM in NPC1 deficient cells was roughly one-tenth of wild-type MEFs, and the improved potency was seen across all LNP doses tested (Fig. 4e). To further measure cellular retention and siRNA efficacy, NPC1 +/+ and NPC1 −/− MEFs (stably transfected with GFP) were treated with LNPs ...
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... or NPC1 −/− MEFs at different concentra- tions. A substantial improvement in LNP potency was observed in NPC1 −/− cells in comparison to wild-type cells (Fig. 4e). The median inhibitory concentration (IC 50 ) of 1.5 nM in NPC1 deficient cells was roughly one-tenth of wild-type MEFs, and the improved potency was seen across all LNP doses tested (Fig. 4e). To further measure cellular retention and siRNA efficacy, NPC1 +/+ and NPC1 −/− MEFs (stably transfected with GFP) were treated with LNPs containing GFP siRNA; high-throughput microscopy was used to simultaneously measure cellular retention and GFP fluorescence 72 h after incubation in both cell types. Enhanced cellular retention of ...
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... , LNPs remain in enlarged late endosomes/lysosomes and are not recycled back out of the cell. One hypothesis to explain how this leads to a greater efficiency in siRNA-mediated gene silenc- ing is that the increased residence time of more siRNA inside cells results in a slow, controlled diffusion of the siRNA into the cytoplasm npg l e t t e r s (Fig. 4f (i,ii)). It appears that late endosome/multivesicular late endo- somes may serve as a transient reservoir where LNP recycle through multiple pathways. Prevention of direct egress of LNPs from the late endosomes leads to enhanced retention, and perinuclear positioning of these vesicular structures may serve as a persistent site for endo- somal ...
Citations
... For instance, Kichler et al. [72] tested the proton sponge hypothesis for PEI-mediated delivery by using proton pump inhibitors, bafilomycin A1, and concanamycin A, to assess any reduction in endosomal escape. The inhibition strategy lends itself well to high-throughput studies, exemplified by Sahay et al.'s screening of a library of small molecule inhibitors in cell culture, where microscopy was employed to identify the effectors necessary for lipid nanoparticle cellular entry [73]. However, it is noteworthy that most inhibitors may exert effects on multiple intracellular processes, thus limiting the specificity of this approach [74]. ...
The delivery of therapeutic agents faces significant hurdles posed by the endo-lysosomal pathway, a bottleneck that hampers clinical effectiveness. This comprehensive review addresses the urgent need to enhance cellular delivery mechanisms to overcome these obstacles. It focuses on the potential of smart nanomaterials, delving into their unique characteristics and mechanisms in detail. Special attention is given to their ability to strategically evade endosomal entrapment, thereby enhancing therapeutic efficacy. The manuscript thoroughly examines assays crucial for understanding endosomal escape and cellular uptake dynamics. By analyzing various assessment methods, we offer nuanced insights into these investigative approaches’ multifaceted aspects. We meticulously analyze the use of smart nanocarriers, exploring diverse mechanisms such as pore formation, proton sponge effects, membrane destabilization, photochemical disruption, and the strategic use of endosomal escape agents. Each mechanism’s effectiveness and potential application in mitigating endosomal entrapment are scrutinized. This paper provides a critical overview of the current landscape, emphasizing the need for advanced delivery systems to navigate the complexities of cellular uptake. Importantly, it underscores the transformative role of smart nanomaterials in revolutionizing cellular delivery strategies, leading to a paradigm shift towards improved therapeutic outcomes.
... 11 For instance: delivery of siRNA and other chemotherapeutics by lipid nanoparticles or EV based therapeutic delivery is limited by endosomal entrapment. 12,13 This unfortunate fate can render potentially life-saving medications ineffective. 12 Consequently, there is an urgent need to develop widely applicable technologies that can modulate and precisely control the trafficking of drug, molecules, and nanoparticles within cells. ...
... 12,13 This unfortunate fate can render potentially life-saving medications ineffective. 12 Consequently, there is an urgent need to develop widely applicable technologies that can modulate and precisely control the trafficking of drug, molecules, and nanoparticles within cells. By unraveling the intricate mechanisms and developing disruptive technological approaches to govern intracellular transport, we can begin to overcome the challenges posed to drug delivery and enhance the efficacy of treatments. ...
Chemotherapy resistance and endosomal entrapment, controlled by intracellular trafficking processes, is a major factor in treatment failure. Here, we test the hypothesis that external electrical stimulus can be used to modulate intracellular trafficking of chemotherapeutic drugs in most common malignant brain tumors in childhood (medulloblastoma) and gold nanoparticles (GNPs) in adulthood (glioblastoma). We demonstrate that application of alternating current (AC) with frequencies ranging from KHz-MHz and low strength (1V/cm) lead to killing of cisplatin and vincristine resistant (mediated by extracellular vesicles) medulloblastoma cell lines. On the other hand, in primary glioblastoma cells, high frequency AC (MHz) regulated the endosomal escape of GNPs. No significant effect on the viability of the control medulloblastoma cells (resistant cells cultured in drug free media and non-resistant cells) and glioblastoma cells after AC treatment confirmed targeting of intracellular trafficking process. This work supports future application of AC in drug delivery and brain cancer therapy.
... In addition, identifying the exact endosomal compartment of escape is also crucial to improve cellular uptake [117] and it still is a debatable aspect. Hence, to understand how the uptake and the transport to endosomal compartments can affect the cargo's delivery and release, nanocarriers with different properties have been investigated [118][119][120]. For instance, Paramasivam et al. compared the uptake and endosomal distribution of six mRNA-LNPs formulations, with similar size and RNA contents, but diverse chemical composition. ...
Breast cancer (BC) prevails as a major burden on global healthcare, being the most prevalent form of cancer among women. BC is a complex and heterogeneous disease, and current therapies, such as chemotherapy and radiotherapy, frequently fall short in providing effective solutions. These treatments fail to mitigate the risk of cancer recurrence and cause severe side effects that, in turn, compromise therapeutic responses in patients. Over the last decade, several strategies have been proposed to overcome these limitations. Among them, RNA-based technologies have demonstrated their potential across various clinical applications, notably in cancer therapy. However, RNA therapies are still limited by a series of critical issues like off-target effect and poor stability in circulation. Thus, novel approaches have been investigated to improve the targeting and bioavailability of RNA-based formulations to achieve an appropriate therapeutic outcome. Lipid nanoparticles (LNPs) have been largely proven to be an advantageous carrier for nucleic acids and RNA. This perspective explores the most recent advances on RNA-based technology with an emphasis on LNPs’ utilization as effective nanocarriers in BC therapy and most recent progresses in their clinical applications.
Graphical Abstract
... Liposomes and liposome-mRNA complexes (lipoplexes) can likely be internalized in multiple ways, including clathrin or caveolin-mediated endocytosis, macropinocytosis, and phagocytosis, as well as by direct fusion with the cell membrane [15,[18][19][20][21]. Typically, the uptaken RNA-LNPs are entrapped in early endosomes and, after they mature to late endosomes and then to endolysosomes, degraded by acidic pH and endolysosomal enzymes [22][23][24][25][26]. Additionally, they can be returned back to the extracellular space via exocytic (recycling) route [26][27][28][29]. ...
... Liposomes and liposome-mRNA complexes (lipoplexes) can likely be internalized in multiple ways, including clathrin or caveolin-mediated endocytosis, macropinocytosis, and phagocytosis, as well as by direct fusion with the cell membrane [15,[18][19][20][21]. Typically, the uptaken RNA-LNPs are entrapped in early endosomes and, after they mature to late endosomes and then to endolysosomes, degraded by acidic pH and endolysosomal enzymes [22][23][24][25][26]. Additionally, they can be returned back to the extracellular space via exocytic (recycling) route [26][27][28][29]. Both of these pathways mark non-productive trafficking. ...
... This brings mRNA to the cytoplasmic translation machinery, thus marking functional delivery of the cargo [17,30]. Yet, endosomal escape occurs with only a small fraction of RNA molecules, representing the bottleneck for efficient transfection [18,[22][23][24][25][26]31]. ...
Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.
... 43 After arriving at the target, LNPs can be internalized through dynamin-dependent pathway, clathrin-dependent pathway, macropinocytosis, or direct fusion with the cell membrane. 44 Eventually the internalization mechanism depends on many factors, including size, ionizable lipid of choice, and helper lipids. 45 Finally, once inside the endosome, the ionizable cationic lipid becomes positively charged due to the low pH in the endosome, and this initiates the destabilization of the endosome with the LNP-endosomal membrane adhesion, fusion, and final disruption facilitated by cholesterol and helper lipids through phase transition of the LNPs ( Figure 6). ...
Locked nucleic acids (LNAs) are a subtype of antisense oligonucleotides (ASOs) that are characterized by a bridge within the sugar moiety. LNAs owe their robustness to this chemical modification, which as the name suggests, locks it in one conformation. This perspective includes two components: a general overview on ASOs from one side and on delivery issues focusing on lipid nanoparticles (LNPs) on the other side. Throughout, a screening of the ongoing clinical trials involving ASOs is given, as well as a take on the versatility and challenges of using LNAs. Finally, we highlight the potential of LNPs as carriers for the successful delivery of LNAs.
... In cells treated with cKK-E12 LNPs, >50% of endosomes are ruptured. Given that it is estimated that ~1% of cargo mRNA escape to the cytosol [45][46][47][48] , the high fraction of damaged endosomes suggests that endosomal escape of RNA comes with a large amount of collateral endosomal damage. ...
Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs’ hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.
... Despite the developments in the LNP field, a commonly overlooked aspect regards the very limited release of the nucleic acid payloads in the cytoplasm (17)(18)(19). LNPs internalize into cells, via both clathrin-dependent and clathrin-independent endocytosis mechanisms such as macropinocytosis (17,20). The majority of the particles are endocytosed by macropinocytosis; however, clathrin-mediated endocytosis is a prerequisite. ...
... This process is inefficient and is considered a bottleneck in this field (17,18). Previous studies showed that the majority of the RNA-LNPs that internalize into target cells are either degraded by lysosomes or recycled outside of the target cells, with only a very limited amount of RNA payloads released into the cytoplasm (17,20). Currently, there are disagreements on the endocytic stage of RNA payload release into the cytosol (17,20,22,23). ...
... Previous studies showed that the majority of the RNA-LNPs that internalize into target cells are either degraded by lysosomes or recycled outside of the target cells, with only a very limited amount of RNA payloads released into the cytoplasm (17,20). Currently, there are disagreements on the endocytic stage of RNA payload release into the cytosol (17,20,22,23). Further complicating the matter, the knowledge gained on siRNA delivery does not necessarily translate to mRNA payload release since several studies suggest differences between the escape of siRNA and mRNA payloads (17,18,22). ...
Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A major bottleneck in the field is the release of mRNA-LNPs from the endosomal pathways into the cytosol of cells where they can execute their encoded functions. The data regarding the mechanism of these endosomal escape processes are limited and contradicting. Despite extensive research, there is no consensus regarding the compartment of escape, the cause of the inefficient escape and are currently lacking a robust method to detect the escape. Here, we review the currently known mechanisms of endosomal escape and the available methods to study this process. We critically discuss the limitations and challenges of these methods and the possibilities to overcome these challenges. We propose that the development of currently lacking robust, quantitative high-throughput techniques to study endosomal escape is timely and essential. A better understanding of this process will enable better RNA-LNP designs with improved efficiency to unlock new therapeutic modalities.
... The cellular destination of LNPs can have a significant impact on the efficacy of gene delivery, due to differences in endo-lysosomal trafficking between cell types and internalization pathways. [27][28][29] To achieve the desired tissue and cellular specificity in vivo, antibodies (and fragments) are often conjugated to the surface of nanoparticles. This strategy, which can enhance accumulation by orders of magnitude in the desired tissue, also has the potential to interfere with the endosomal escape of mRNA that was optimized for an uncoated particle. ...
RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic (PK)-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically based PK/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles (LNPs) in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue PKs of LNPs, as well as the kinetics of tissue luciferase expression measured by ex vivo activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within a 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the in vivo behavior of RNA-containing LNPs developed for an array of conditions and across species.
... [5a,b,17] However, the cholesterol transporter on the surface of late endosome causes the efflux of LNPs with high amounts of cholesterol thus reducing LNPs' efficacy. [18] To overcome this limitation, cholesterol derivatives have been developed that can modulate the endosomal trafficking of LNPs by reducing the release of endocytosed LNPs into the extracellular environment. [16a] Histidine is a well-known amino acid that enhances the endosomal escape of nanoparticles by protonation in an acidic environment. ...
Recently, mRNA‐based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3β[L‐histidinamide‐carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pKa values of the Hchol‐LNPs are ≈6.03 and 6.61 in MC3‐ and SM102‐based lipid formulations. Notably, the Hchol‐LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol‐LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol‐LNPs. Importantly, LNP‐encapsulated SARS‐CoV‐2 spike mRNA vaccinations induce potent antigen‐specific antibodies against SARS‐CoV‐2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.
... One of the key issues to fully realize the therapeutic potential of proteins in cells is the delivery of active proteins to the cytosol. [2] Currently, membrane-permeable nanocarriers, such as liposomes, [3] lipid nanoparticles, [4] polymers, [5] and inorganic nanoparticles [6] can increase the specific cellular uptake of proteins; however, only a small fraction (<10%) of proteins reach the cytosol [7] due to exocytosis [8] and endocytic sequestration. Moreover, the bioactivity of proteins can be compromised by endosomal-lysosomal degradation. ...
Despite the potential of protein therapeutics, the cytosolic delivery of proteins with high efficiency and bioactivity remains a significant challenge owing to exocytosis and lysosomal degradation after endocytosis. Therefore, it is important to develop a safe and efficient strategy to bypass endocytosis. Inspired by the extraordinary capability of filamentous‐actin (F‐actin) to promote cell membrane fusion, a cyanine dye assembly‐containing nanoplatform mimicking the structure of natural F‐actin is developed. The nanoplatform exhibits fast membrane fusion to cell membrane mimics and thus enters live cells through membrane fusion and bypasses endocytosis. Moreover, it is found to efficiently deliver protein cargos into live cells and quickly release them into the cytosol, leading to high protein cargo transfection efficiency and bioactivity. The nanoplatform also results in the superior inhibition of tumor cells when loaded with anti‐tumor proteins. These results demonstrate that this fusogenic nanoplatform can be valuable for cytosolic protein delivery and tumor treatment.
