Light triggered nanoscale biolistics for efficient intracellular delivery of functional macromolecules in mammalian cells.

Biolistic intracellular delivery of functional macromolecules makes use of dense microparticles which are ballistically fired onto cells with a pressurized gun. While it has been used to transfect plant cells, its application to mammalian cells has met with limited success mainly due to high toxicity. Here we present a more refined nanotechnological approach to biolistic delivery with light-triggered self-assembled nanobombs (NBs) that consist of a photothermal core particle surrounded by smaller nanoprojectiles. Upon irradiation with pulsed laser light, fast heating of the core particle results in vapor bubble formation, which propels the nanoprojectiles through the cell membrane of nearby cells. We show successful transfection of both adherent and non-adherent cells with mRNA and pDNA, outperforming electroporation as the most used physical transfection technology by a factor of 5.5-7.6 in transfection yield. With a throughput of 104-105 cells per second, biolistic delivery with NBs offers scalable and highly efficient transfections of mammalian cells.

Black phosphorus mediated photoporation: a broad absorption nanoplatform for intracellular delivery of macromolecules.

Nanoparticle-sensitized photoporation for intracellular delivery of external compounds usually relies on the use of spherical gold nanoparticles as sensitizing nanoparticles. As they need stimulation with visible laser light, they are less suited for transfection of cells in thick biological tissues. In this work, we have explored black phosphorus quantum dots (BPQDs) as alternative sensitizing nanoparticles for photoporation with a broad and uniform absorption spectrum from the visible to the near infra-red (NIR) range. We demonstrate that BPQD sensitized photoporation allows efficient intracellular delivery of both siRNA (>80%) and mRNA (>40%) in adherent cells as well as in suspension cells. Cell viability remained high (>80%) irrespective of whether irradiation was performed with visible (532 nm) or near infrared (800 nm) pulsed laser light. Finally, as a proof of concept, we used BPQD sensitized photoporation to deliver macromolecules in cells with thick phantom tissue in the optical path. NIR laser irradiation resulted in only 1.3× reduction in delivery efficiency as compared to photoporation without the phantom gel, while with visible laser light the delivery efficiency was reduced 2×.

Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering.

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

Bubble Forming Films for Spatial Selective Cell Killing.

Photodynamic and photothermal cell killing at the surface of tissues finds applications in medicine. However, a lack of control over heat dissipation following a treatment with light might damage surrounding tissues. A new strategy to kill cells at the surface of tissues is reported. Polymeric films are designed in which iron oxide nanoparticles are embedded as photosensitizers. Irradiation of the films with pulsed laser light generates water vapor bubbles at the surface of the films. It is found that "bubble-films" can kill cells in close proximity to the films due to mechanical forces which arise when the bubbles collapse. Local irradiation of bubble-films allows for spatial selective single cell killing. As nanosurgery becomes attractive in ophthalmology to remove superficial tumors, bubble-films are applied on the cornea and it is found that irradiation of the bubble-films allows spatial and selective killing of corneal cells. As i) the photosensitizer is embedded in the films, which reduces its uptake by cells and spreading into tissues and ii) the bubble-films can be removed from the tissue after laser treatment, while iii) a low laser fluence is sufficient to generate vapor bubbles, it is foreseen that bubble-films might become promising for safe resection of superficial tumors.

Nanoparticle-sensitized photoporation enables inflammasome activation studies in targeted single cells.

Inflammasomes are multi-protein complexes that guard against cellular stress and microbial infections. Inflammasome activation studies frequently require delivery of pathogen-derived virulence factors into the cytosol of macrophages and other innate immune cells. This is a challenging requirement since primary macrophages are difficult-to-transfect, especially when it comes to the intracellular delivery of proteins. Here, we report on the use of nanoparticle-sensitized photoporation as a promising upcoming intracellular delivery technology for delivering proteins of various molecular weights into the cytosol of primary macrophages. While 60-70 nm gold nanoparticles are the most commonly used sensitizing nanoparticles for photoporation, here we find that 0.5 µm iron oxide nanoparticles perform markedly better on primary macrophages. We demonstrate that LFn-FlaA or lipopolysaccharides can be delivered in primary macrophages resulting in activation of the NLRC4 or the non-canonical inflammasome, respectively. We furthermore show that photoporation can be used for targeted delivery of these toxins into selected cells, opening up the possibility to study the interaction between inflammasome activated cells and surrounding healthy cells. Taken together, these results show that nanoparticle-sensitized photoporation is very well suited to deliver pathogenic virulence factors in primary macrophages, thus constituting an effective new enabling technology for inflammasome activation studies.

Cytosolic delivery of gadolinium via photoporation enables improved in vivo magnetic resonance imaging of cancer cells.

Longitudinal in vivo monitoring of transplanted cells is crucial to perform cancer research or to assess the treatment outcome of cell-based therapies. While several bio-imaging techniques can be used, magnetic resonance imaging (MRI) clearly stands out in terms of high spatial resolution and excellent soft-tissue contrast. However, MRI suffers from low sensitivity, requiring cells to be labeled with high concentrations of contrast agents. An interesting option is to label cells with clinically approved gadolinium chelates which generate a hyperintense MR signal. However, spontaneous uptake of the label via pinocytosis results in its endosomal sequestration, leading to quenching of the T1-weighted relaxation. To avoid this quenching effect, delivery of gadolinium chelates directly into the cytosol via electroporation or hypotonic cell swelling have been proposed. However, these methods are also accompanied by several drawbacks such as a high cytotoxicity, and changes in gene expression and phenotype. Here, we demonstrate that nanoparticle-sensitized laser induced photoporation forms an attractive alternative to efficiently deliver the contrast agent gadobutrol into the cytosol of both HeLa and SK-OV-3 IP1 cells. After intracellular delivery by photoporation the quenching effect is clearly avoided, leading to a strong increase in the hyperintense T1-weighted MR signal. Moreover, when compared to nucleofection as a state-of-the-art electroporation platform, photoporation has much less impact on cell viability, which is extremely important for reliable cell tracking studies. Additional experiments confirm that photoporation does not induce any change in the long-term viability or the migratory capacity of the cells. Finally, we show that gadolinium 'labeled' SK-OV-3 IP1 cells can be imaged in vivo by MRI with high soft-tissue contrast and spatial resolution, revealing indications of potential tumor invasion or angiogenesis.

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation.

Fluorescence microscopy is the method of choice for studying intracellular dynamics. However, its success depends on the availability of specific and stable markers. A prominent example of markers that are rapidly gaining interest are nanobodies (Nbs, ~ 15 kDa), which can be functionalized with bright and photostable organic fluorophores. Due to their relatively small size and high specificity, Nbs offer great potential for high-quality long-term subcellular imaging, but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells. We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability. Being a laser-based technology, it is readily compatible with light microscopy and the typical cell recipients used for that. Spurred by these promising initial results, we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells. We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm, actin-bundling protein Fascin, and the histone H2A/H2B heterodimers. With an efficiency of more than 80% labeled cells and minimal toxicity (~ 2%), photoporation proved to be an excellent intracellular delivery method for Nbs. Time-lapse microscopy revealed that cell division rate and migration remained unaffected, confirming excellent cell viability and functionality. We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells, laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

Surface Functionalization with Polyethylene Glycol and Polyethyleneimine Improves the Performance of Graphene-Based Materials for Safe and Efficient Intracellular Delivery by Laser-Induced Photoporation.

Nanoparticle mediated laser-induced photoporation is a physical cell membrane disruption approach to directly deliver extrinsic molecules into living cells, which is particularly promising in applications for both adherent and suspension cells. In this work, we explored surface modifications of graphene quantum dots (GQD) and reduced graphene oxide (rGO) with polyethylene glycol (PEG) and polyethyleneimine (PEI) to enhance colloidal stability while retaining photoporation functionality. After photoporation with FITC-dextran 10 kDa (FD10), the percentage of positive HeLa cells (81% for GQD-PEG, 74% for rGO-PEG and 90% for rGO-PEI) increased approximately two-fold compared to the bare nanomaterials. While for Jurkat suspension cells, the photoporation efficiency with polymer-modified graphene-based nanomaterial reached as high as 80%. Cell viability was >80% in all these cases. In addition, polymer functionalization proved to be beneficial for the delivery of larger macromolecules (FD70 and FD500) as well. Finally, we show that rGO is suitable for photoporation using a near-infrared laser to reach 80% FD10 positive HeLa cells at 80% cell viability. We conclude that modification of graphene-based nanoparticles with PEG and especially PEI provide better colloidal stability in cell medium, resulting in more uniform transfection and overall increased efficiency.

Vapor nanobubble is the more reliable photothermal mechanism for inducing endosomal escape of siRNA without disturbing cell homeostasis.

Strategies for controlled delivery of therapeutic siRNA into living cells are in high demand as endosomal escape remains the most prominent bottleneck at the intracellular level. Photothermal properties of gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation by two mechanisms: endosomal rupture by mechanical energy from water vapor nanobubbles (VNBs), or permeabilization of the endosomal membrane by heat diffusion. Here we evaluated how both mechanisms influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. Using a siRNA/AuNP drug delivery system we found that the in vitro release of siRNA from the AuNP carrier occurs equally efficiently by VNB formation or heat generation. Heat-mediated endosomal escape happened more efficiently in cells that had more particles per endosome, resulting in variable siRNA-induced downregulation (20-50%). VNB-mediated endosomal escape did not dependent on the number of AuNP per endosome, yielding high downregulations (50-60%) independent of the cell type. Effects on cell homeostasis by whole transcriptome analysis, showed a quick recover after 24 h or 48 h for either of both photothermal mechanisms. We conclude that VNBs are more consistent to induce efficient endosomal escape and gene silencing independent of the cell type without long lasting effects on cell homeostasis.

Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation.

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications, such as CAR T cell-based therapies. mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral- or DNA transposon-based approaches for the generation of adoptive T cells. However, limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells. In this study, we demonstrate that gold nanoparticle-mediated vapor nanobubble (VNB) photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells. Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration, while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%. Importantly, compared to electroporation, which is the reference technology for nonviral transfection of T cells, a fivefold increase in the number of transfected viable Jurkat T cells was observed. Altogether, our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells, with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells.

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism.

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed 'sonoprinting'. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells.

Photoablation of Human Vitreous Opacities by Light-Induced Vapor Nanobubbles.

Myopia, diabetes, and aging are the main causes of progressive vitreous collagen aggregation, resulting in vitreous opacities, which can significantly disturb vision. As vitreous opacities, which induce the visual phenomenon of "floaters", are accessible with nanomaterials and light, we propose a nanotechnology-based approach to locally ablate them with highly reduced light energy compared to the more traditional YAG laser therapy. Our strategy relies on the plasmon properties of gold nanoparticles that generate vapor nanobubbles upon pulsed-laser illumination whose mechanical force can ablate vitreous opacities. We designed gold nanoparticles coated with hyaluronic acid (HA), which have excellent diffusional mobility in human vitreous, an essential requirement to reach the vitreous opacities. In addition, we found that HA-coated gold nanoparticles can accumulate extensively on human vitreous opacities that were obtained by vitrectomy from patients with vision-degrading myodesopsia. When subsequently applying nanosecond laser pulses, the collagen aggregates were efficiently destroyed with ∼1000 times less light energy than typically used in YAG laser therapy. This low-energy "floater-specific destruction", which is due to the accumulation of the small gold nanoparticles on the opacities, is attractive, as it may be safer to the surrounding ocular tissues while at the same time being easier and faster to apply compared to YAG laser therapy, where the opacities need to be ablated piece by piece by a tightly focused laser beam. Gold nanoparticle-assisted photoablation may therefore provide a safer, faster, and more reliable destruction of vitreous opacities in the treatment of ophthalmologic diseases.

Laser-induced vapour nanobubbles improve drug diffusion and efficiency in bacterial biofilms.

Hindered penetration of antibiotics through biofilms is one of the reasons for the alarming increase in bacterial tolerance to antibiotics. Here, we investigate the potential of laser-induced vapour nanobubbles (VNBs) formed around plasmonic nanoparticles to locally disturb biofilm integrity and improve antibiotics diffusion. Our results show that biofilms of both Gram-negative (Burkholderia multivorans, Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria can be loaded with cationic 70-nm gold nanoparticles and that subsequent laser illumination results in VNB formation inside the biofilms. In all types of biofilms tested, VNB formation leads to substantial local biofilm disruption, increasing tobramycin efficacy up to 1-3 orders of magnitude depending on the organism and treatment conditions. Altogether, our results support the potential of laser-induced VNBs as a new approach to disrupt biofilms of a broad range of organisms, resulting in improved antibiotic diffusion and more effective biofilm eradication.

Targeted Perturbation of Nuclear Envelope Integrity with Vapor Nanobubble-Mediated Photoporation.

The nuclear envelope (NE) has long been considered to dismantle only during mitosis. However, recent observations in cancer cells and laminopathy patient cells have revealed that the NE can also transiently rupture during interphase, thereby perturbing cellular homeostasis. Although NE ruptures are promoted by mechanical force and the loss of lamins, their stochastic nature and variable frequency preclude the study of their direct downstream consequences. We have developed a method based on vapor nanobubble-mediated photoporation that allows for deliberately inducing NE ruptures in a spatiotemporally controlled manner. Our method relies on wide-field laser illumination of perinuclear gold nanoparticles, resulting in the formation of short-lived vapor nanobubbles that inflict minute mechanical damage to the NE, thus creating small pores. We demonstrate that perinuclear localization of gold nanoparticles can be achieved after endocytic uptake or electroporation-facilitated delivery and that both strategies result in NE rupture upon laser irradiation. Furthermore, we prove that photoporation-induced nuclear ruptures are transient and recapitulate hallmarks of spontaneous NE ruptures that occur in A-type lamin-depleted cells. Finally, we show that the same approach can be used to promote influx of macromolecules that are too large to passively migrate through the NE. Thus, by providing unprecedented control over nuclear compartmentalization, nuclear photoporation offers a powerful tool for both fundamental cell biology research and drug delivery applications.

Endosomal Size and Membrane Leakiness Influence Proton Sponge-Based Rupture of Endosomal Vesicles.

In gene therapy, endosomal escape represents a major bottleneck since nanoparticles often remain entrapped inside endosomes and are trafficked toward the lysosomes for degradation. A detailed understanding of the endosomal barrier would be beneficial for developing rational strategies to improve transfection and endosomal escape. By visualizing individual endosomal escape events in live cells, we obtain insight into mechanistic factors that influence proton sponge-based endosomal escape. In a comparative study, we found that HeLa cells treated with JetPEI/pDNA polyplexes have a 3.5-fold increased endosomal escape frequency compared to ARPE-19 cells. We found that endosomal size has a major impact on the escape capacity. The smaller HeLa endosomes are more easily ruptured by the proton sponge effect than the larger ARPE-19 endosomes, a finding supported by a mathematical model based on the underlying physical principles. Still, it remains intriguing that even in the small HeLa endosomes, <10% of the polyplex-containing endosomes show endosomal escape. Further experiments revealed that the membrane of polyplex-containing endosomes becomes leaky to small compounds, preventing effective buildup of osmotic pressure, which in turn prevents endosomal rupture. Analysis of H1299 and A549 cells revealed that endosomal size determines endosomal escape efficiency when cells have comparable membrane leakiness. However, at high levels of membrane leakiness, buildup of osmotic pressure is no longer possible, regardless of endosomal size. Based on our findings that both endosomal size and membrane leakiness have a high impact on proton sponge-based endosomal rupture, we provide important clues toward further improvement of this escape strategy.

Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy.

In the replacement of genetic probes, there is increasing interest in labeling living cells with high-quality extrinsic labels, which avoid over-expression artifacts and are available in a wide spectral range. This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells. Here, we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique. We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation. We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses, allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells. We demonstrate high-quality time-lapse imaging with confocal, total internal reflection fluorescence (TIRF), and Airyscan super-resolution microscopy. As the entire procedure is readily compatible with fluorescence (super resolution) microscopy, photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.

Nucleic acid loading and fluorescent labeling of isolated extracellular vesicles requires adequate purification.

Extracellular vesicles (EVs) are nanosized vesicular structures released by cells to communicate with one another. The growing interest in the (patho)physiological function and potential pharmaceutical application of these vesicles is accompanied by a vast number of new research groups entering this research field and a plethora of different protocols to separate EVs from non-vesicular components. This lack of uniformity often generates conflicting or difficult-to-compare results. Here we provide a comparative analysis of different EV isolation strategies, discussing the purity of the final isolate and highlighting the importance of purity on downstream experimental readouts. First, we show that ultracentrifugation (UC) of B16F10 melanoma cell-derived conditioned medium co-purifies proteins or protein complexes with nuclease activity. Such contaminants should be taken into account when aiming to apply EVs as delivery carriers for exogenous nucleic acids. Second, three commonly used purification strategies (i.e. precipitation, UC and density-gradient centrifugation) were evaluated for their ability to remove non-incorporated fluorescent dye (i.e. the lipophilic PKH67 dye), important when probing EV interactions with cells. For both types of impurities, endogenous and exogenous, density gradient purification outperforms the other evaluated methods. Overall, these results demonstrate that the implementation of stringent purification protocols and adequate controls is of pivotal importance to draw reliable conclusions from downstream experiments performed with EV isolates.

Intracellular delivery of oligonucleotides in Helicobacter pylori by fusogenic liposomes in the presence of gastric mucus.

The rising antimicrobial resistance contributes to 25000 annual deaths in Europe. This threat to the public health can only be tackled if novel antimicrobials are developed, combined with a more precise use of the currently available antibiotics through the implementation of fast, specific, diagnostic methods. Nucleic acid mimics (NAMs) that are able to hybridize intracellular bacterial RNA have the potential to become such a new class of antimicrobials and additionally could serve as specific detection probes. However, an essential requirement is that these NAMs should be delivered into the bacterial cytoplasm, which is a particular challenge given the fact that they are charged macromolecules. We consider these delivery challenges in relation to the gastric pathogen Helicobacter pylori, the most frequent chronic infection worldwide. In particular, we evaluate if cationic fusogenic liposomes are suitable carriers to deliver NAMs across the gastric mucus barrier and the bacterial envelope. Our study shows that DOTAP-DOPE liposomes post-PEGylated with DSPE-PEG (DSPE Lpx) can indeed successfully deliver NAMs into Helicobacter pylori, while offering protection to the NAMs from binding and inactivation in gastric mucus isolated from pigs. DSPE Lpx thus offer exciting new possibilities for in vivo diagnosis and treatment of Helicobacter pylori infections.