Enhancing the In Vitro and In Vivo Stabilities of Polymeric Nucleic Acid Delivery Nanosystems.

Gene therapy holds great promise for various medical and biomedical applications. Nonviral gene delivery systems formed by cationic polymer and nucleic acids (e.g., polyplexes) have been extensively investigated for targeted gene therapy; however, their in vitro and in vivo stability is affected by both their intrinsic properties such as chemical compositions (e.g., polymer molecular weight and structure, and N/P ratio) and a number of environmental factors (e.g., shear stress during circulation in the bloodstream, interaction with the serum proteins, and physiological ionic strength). In this review, we surveyed the effects of a number of important intrinsic and environmental factors on the stability of polymeric gene delivery systems, and discussed various strategies to enhance the stability of polymeric gene delivery systems, thereby enabling efficient gene delivery into target cells. Future opportunities and challenges of polymeric nucleic acid delivery nanosystems were also briefly discussed.

Unimolecular Micelle-Based Hybrid System for Perivascular Drug Delivery Produces Long-Term Efficacy for Neointima Attenuation in Rats.

At present, there are no clinical options for preventing neointima-caused (re)stenosis after open surgery such as bypass surgery for treating flow-limiting vascular disease. Perivascular drug delivery is a promising strategy, but in translational research, it remains a major challenge to achieve long-term (e.g., > 3 months) anti(re)stenotic efficacy. In this study, we engineered a unique drug delivery system consisting of durable unimolecular micelles, formed by single multiarm star amphiphilic block copolymers with only covalent bonds, and a thermosensitive hydrogel formed by a poly(lactide-co-glycolide)-poly(ethylene glycol)-poly(lactide-co-glycolide) triblock copolymer (abbreviated as triblock gel) that is stable for about 4 weeks in vitro. The drug-containing unimolecular micelles (UMs) suspended in Triblock gel were able to sustain rapamycin release for over 4 months. Remarkably, even 3 months after perivascular application of the rapamycin-loaded micelles in Triblock gel in the rat model, the intimal/medial area ratio (a restenosis measure) was still 80% inhibited compared to the control treated with empty micelle/gel (no drug). This could not be achieved by applying rapamycin in Triblock gel alone, which reduced the intimal/medial ratio only by 27%. In summary, we created a new UM/Triblock gel hybrid system for perivascular drug delivery, which produced a rare feat of 3-month restenosis inhibition in animal tests. This system exhibits a real potential for further translation into an anti(re)stenotic application with open surgery.

Photodynamic Therapy for Atherosclerosis: Past, Present, and Future.

This review paper examines the evolution of photodynamic therapy (PDT) as a novel, minimally invasive strategy for treating atherosclerosis, a leading global health concern. Atherosclerosis is characterized by the accumulation of lipids and inflammation within arterial walls, leading to significant morbidity and mortality through cardiovascular diseases such as myocardial infarction and stroke. Traditional therapeutic approaches have primarily focused on modulating risk factors such as hypertension and hyperlipidemia, with emerging evidence highlighting the pivotal role of inflammation. PDT, leveraging a photosensitizer, specific-wavelength light, and oxygen, offers targeted treatment by inducing cell death in diseased tissues while sparing healthy ones. This specificity, combined with advancements in nanoparticle technology for improved delivery, positions PDT as a promising alternative to traditional interventions. The review explores the mechanistic basis of PDT, its efficacy in preclinical studies, and the potential for enhancing plaque stability and reducing macrophage density within plaques. It also addresses the need for further research to optimize treatment parameters, mitigate adverse effects, and validate long-term outcomes. By detailing past developments, current progress, and future directions, this paper aims to highlight PDT's potential in revolutionizing atherosclerosis treatment, bridging the gap from experimental research to clinical application.

Multimodal nanoimmunotherapy engages neutrophils to eliminate Staphylococcus aureus infections.

The increasing prevalence of antimicrobial resistance in Staphylococcus aureus necessitates alternative therapeutic approaches. Neutrophils play a crucial role in the fight against S. aureus but suffer from deficiencies in function leading to increased infection. Here we report a nanoparticle-mediated immunotherapy aimed at potentiating neutrophils to eliminate S. aureus. The nanoparticles consist of naftifine, haemoglobin (Hb) and a red blood cell membrane coating. Naftifine disrupts staphyloxanthin biosynthesis, Hb reduces bacterial hydrogen sulfide levels and the red blood cell membrane modifies bacterial lipid composition. Collectively, the nanoparticles can sensitize S. aureus to host oxidant killing. Furthermore, in the infectious microenvironment, Hb triggers lipid peroxidation in S. aureus, promoting neutrophil chemotaxis. Oxygen supplied by Hb can also significantly enhance the bactericidal capability of the recruited neutrophils by restoring neutrophil respiratory burst via hypoxia relief. This multimodal nanoimmunotherapy demonstrates excellent therapeutic efficacy in treating antimicrobial-resistant S. aureus persisters, biofilms and S. aureus-induced infection in mice.

Targeted NAD+ repletion via biomimetic nanoparticle enables simultaneous management of intimal hyperplasia and accelerated re-endothelialization: A proof-of-concept study toward next-generation of endothelium-protective, anti-restenotic therapy.

Endovascular interventions often fail due to restenosis, primarily caused by smooth muscle cell (SMC) proliferation, leading to intimal hyperplasia (IH). Current strategies to prevent restenosis are far from perfect and impose significant collateral damage on the fragile endothelial cell (EC), causing profound thrombotic risks. Nicotinamide adenine dinucleotide (NAD+) is a co-enzyme and signaling substrate implicated in redox and metabolic homeostasis, with a pleiotropic role in protecting against cardiovascular diseases. However, a functional link between NAD+ repletion and the delicate duo of IH and EC regeneration has yet to be established. NAD+ repletion has been historically challenging due to its poor cellular uptake and low bioavailability. We have recently invented the first nanocarrier that enables direct intracellular delivery of NAD+ in vivo. Combining the merits of this prototypic NAD+-loaded calcium phosphate (CaP) nanoparticle (NP) and biomimetic surface functionalization, we created a biomimetic P-NAD+-NP with platelet membrane coating, which enabled an injectable modality that targets IH with excellent biocompatibility. Using human cell primary culture, we demonstrated the benefits of NP-assisted NAD+ repletion in selectively inhibiting the excessive proliferation of aortic SMC, while differentially protecting aortic EC from apoptosis. Moreover, in a rat balloon angioplasty model, a single-dose treatment with intravenously injected P-NAD+-NP immediately post angioplasty not only mitigated IH, but also accelerated the regeneration of EC (re-endothelialization) in vivo in comparison to control groups (i.e., saline, free NAD+ solution, empty CaP-NP). Collectively, our current study provides proof-of-concept evidence supporting the role of targeted NAD+ repletion nanotherapy in managing restenosis and improving reendothelialization.

Overcoming the Blood-Brain Barrier for Gene Therapy via Systemic Administration of GSH-Responsive Silica Nanocapsules.

CRISPR genome editing can potentially treat the root causes of many genetic diseases, including central nervous system (CNS) disorders. However, the promise of brain-targeted therapeutic genome editing relies on the efficient delivery of biologics bypassing the blood-brain barrier (BBB), which represents a major challenge in the development of CRISPR therapeutics. We created and screened a library of glutathione (GSH)-responsive silica nanocapsules (SNCs) for brain targeted delivery of biologics via systemic administration. In vivo studies demonstrate that systemically delivered SNCs conjugated with glucose and rabies virus glycoprotein peptide under glycemic control can efficiently bypass the intact BBB, enabling brain-wide delivery of various biologics including CRISPR genome editors targeting different genes in both Ai14 reporter mice and wild-type mice. In particular, up to 28% neuron editing via systemic delivery of Cre mRNA in Ai14 mice, up to 6.1% amyloid precursor protein (App) gene editing (resulting in 19.1% reduction in the expression level of intact APP), and up to 3.9% tyrosine hydroxylase (Th) gene editing (resulting in 30.3% reduction in the expression level of TH) in wild-type mice are observed. This versatile SNC nanoplatform may offer a novel strategy for the treatment of CNS disorders including Alzheimer's, Parkinson's, and Huntington's disease.

Guanidinium-Rich Lipopeptide-Based Nanoparticle Enables Efficient Gene Editing in Skeletal Muscles.

Genome editing mediated by the CRISPR-Cas system holds great promise for the treatment of genetic diseases. However, safe and efficient in vivo delivery of CRISPR genome editing machinery remains a challenge. Here, we report a lipopeptide-based nanoparticle (LNP) that can efficiently deliver the CRISPR Cas9/sgRNA ribonucleoprotein (RNP) and enable efficient genome editing both in vitro and in vivo. An artificial lipopeptide, GD-LP, was constructed by linking a hydrophilic guanidinium-rich head to an oleic acid-based hydrophobic tail via a disulfide bond. LNP formed by the self-assembly of GD-LP can easily form a complex with RNP with a loading content of up to 20 wt %. The resulting RNP-LNP nanocomplex led to 72.6% gene editing efficiency in GFP-HEK cells with negligible cytotoxicity. The LNP also showed significantly higher transfection efficiencies than Lipofectamine 2000 for the delivery of mRNA in NIH 3T3 and RAW 264.7 and the delivery of plasmid DNA in B78 cells. In vivo studies showed that intramuscular injection of the RNP-LNP nanocomplex in Ai14 mice induced efficient gene editing in muscular tissues. Moreover, the delivery of Cas9 RNP and donor DNA by LNP (i.e., RNP/ssODN-LNP nanocomplex) restored dystrophin expression, reduced skeletal muscle fibrosis, and significantly improved muscle strength in a Duchenne muscular dystrophy (DMD) mouse model.

Non-viral approaches for gene therapy and therapeutic genome editing across the blood-brain barrier.

The success of brain-targeted gene therapy and therapeutic genome editing hinges on the efficient delivery of biologics bypassing the blood-brain barrier (BBB), which presents a significant challenge in the development of treatments for central nervous system disorders. This is particularly the case for nucleic acids and genome editors that are naturally excluded by the BBB and have poor chemical stability in the bloodstream and poor cellular uptake capability, thereby requiring judiciously designed nanovectors administered systemically for intracellular delivery to brain cells such as neurons. To overcome this obstacle, various strategies for bypassing the BBB have been developed in recent years to deliver biologics to the brain via intravenous administration using non-viral vectors. This review summarizes various brain targeting strategies and recent representative reports on brain-targeted non-viral delivery systems that allow gene therapy and therapeutic genome editing via intravenous administration, and highlights ongoing challenges and future perspectives for systemic delivery of biologics to the brain via non-viral vectors.

Neointima abating and endothelium preserving - An adventitia-localized nanoformulation to inhibit the epigenetic writer DOT1L.

Open vascular reconstructions such as bypass are common treatments for cardiovascular disease. Unfortunately, neointimal hyperplasia (IH) follows, leading to treatment failure for which there is no approved therapy. Here we combined the strengths of tailoring nanoplatforms for open vascular reconstructions and targeting new epigenetic mechanisms. We produced adhesive nanoparticles (ahNP) that could be pen-brushed and immobilized on the adventitia to sustainably release pinometostat, an inhibitor drug selective to the epigenetic writer DOT1L that catalyzes histone-3 lysine-79 dimethylation (H3K79me2). This treatment not only reduced IH by 76.8% in injured arteries mimicking open reconstructions in obese Zucker rats with human-like diseases but also avoided the shortcoming of endothelial impairment in IH management. In mechanistic studies, chromatin immunoprecipitation (ChIP) sequencing revealed co-enrichment of the histone mark H3K27ac(acetyl) and its reader BRD4 at the gene of aurora kinase B (AURKB), where H3K79me2 was also enriched as indicated by ChIP-qPCR. Accordingly, DOT1L co-immunoprecipitated with H3K27ac. Furthermore, the known IH driver BRD4 governed the expression of DOT1L which controlled AURKB's protein level, revealing a BRD4- > DOT1L- > AURKB axis. Consistently, AURKB-selective inhibition reduced IH. Thus, this study presents a prototype nanoformulation suited for open vascular reconstructions, and the new insights into chromatin modulators may aid future translational advances.

Micromolded honeycomb scaffold design to support the generation of a bilayered RPE and photoreceptor cell construct.

Age-related macular degeneration (AMD) causes blindness due to loss of retinal pigment epithelium (RPE) and photoreceptors (PRs), which comprise the two outermost layers of the retina. Given the small size of the macula and the importance of direct contact between RPE and PRs, the use of scaffolds for targeted reconstruction of the outer retina in later stage AMD and other macular dystrophies is particularly attractive. We developed microfabricated, honeycomb-patterned, biodegradable poly(glycerol sebacate) (PGS) scaffolds to deliver organized, adjacent layers of RPE and PRs to the subretinal space. Furthermore, an optimized process was developed to photocure PGS, shortening scaffold production time from days to minutes. The resulting scaffolds robustly supported the seeding of human pluripotent stem cell-derived RPE and PRs, either separately or as a dual cell-layered construct. These advanced, economical, and versatile scaffolds can accelerate retinal cell transplantation efforts and benefit patients with AMD and other retinal degenerative diseases.

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo.

Targeted PERK inhibition with biomimetic nanoclusters confers preventative and interventional benefits to elastase-induced abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a progressive aortic dilatation, causing ∼80% mortality upon rupture. Currently, there is no approved drug therapy for AAA. Surgical repairs are invasive and risky and thus not recommended to patients with small AAAs which, however, account for ∼90% of the newly diagnosed cases. It is therefore a compelling unmet clinical need to discover effective non-invasive strategies to prevent or slow down AAA progression. We contend that the first AAA drug therapy will only arise through discoveries of both effective drug targets and innovative delivery methods. There is substantial evidence that degenerative smooth muscle cells (SMCs) orchestrate AAA pathogenesis and progression. In this study, we made an exciting finding that PERK, the endoplasmic reticulum (ER) stress Protein Kinase R-like ER Kinase, is a potent driver of SMC degeneration and hence a potential therapeutic target. Indeed, local knockdown of PERK in elastase-challenged aorta significantly attenuated AAA lesions in vivo. In parallel, we also conceived a biomimetic nanocluster (NC) design uniquely tailored to AAA-targeting drug delivery. This NC demonstrated excellent AAA homing via a platelet-derived biomembrane coating; and when loaded with a selective PERK inhibitor (PERKi, GSK2656157), the NC therapy conferred remarkable benefits in both preventing aneurysm development and halting the progression of pre-existing aneurysmal lesions in two distinct rodent models of AAA. In summary, our current study not only establishes a new intervention target for mitigating SMC degeneration and aneurysmal pathogenesis, but also provides a powerful tool to facilitate the development of effective drug therapy of AAA.

pH-Responsive Polymer Nanoparticles for Efficient Delivery of Cas9 Ribonucleoprotein With or Without Donor DNA.

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) may offer new therapeutics for genetic diseases through gene disruption via nonhomologous end joining (NHEJ) or gene correction via homology-directed repair (HDR). However, clinical translation of CRISPR technology is limited by the lack of safe and efficient delivery systems. Here, facilely fabricated pH-responsive polymer nanoparticles capable of safely and efficiently delivering Cas9 ribonucleoprotein alone (termed NHEJ-NP, diameter = 29.4 nm), or together with donor DNA (termed HDR-NP, diameter = 33.3 nm) are reported. Moreover, intravenously, intratracheally, and intramuscularly injected NHEJ-NP induces efficient gene editing in mouse liver, lung, and skeletal muscle, respectively. Intramuscularly injected HDR-NP also leads to muscle strength recovery in a Duchenne muscular dystrophy mouse model. NHEJ-NP and HDR-NP possess many desirable properties including high payload loading content, small and uniform sizes, high editing efficiency, good biocompatibility, low immunogenicity, and ease of production, storage, and transport, making them great interest for various genome editing applications with clinical potentials.

NAD(H)-loaded nanoparticles for efficient sepsis therapy via modulating immune and vascular homeostasis.

Sepsis is a life-threatening organ dysfunction responsible for nearly 270,000 deaths annually in the United States alone. Nicotinamide adenine dinucleotide (NAD+), an immunomodulator, can potentially treat sepsis; however, clinical application of NAD+ is hindered by its inability to be directly taken up by cells. To address this challenge, a family of nanoparticles (NPs) loaded with either NAD+ or the reduced form of NAD+ (NADH), hereafter NAD(H)-loaded NPs, were engineered to enable direct cellular transport and replenishment of NAD(H). The NAD(H)-loaded NPs improved cellular energy supply, suppressed inflammation and prevented inflammation-induced cell pyroptosis and apoptosis. Therefore, the NPs can help maintain immune homoeostasis and vascular function, two key factors in the pathogenesis of sepsis. The NAD(H)-loaded NPs demonstrated excellent therapeutic efficacies in treating endotoxemia and multidrug-resistant pathogen-induced bacteremia. In addition, the NAD(H)-loaded NPs prevented caecal ligation and puncture-induced multiorgan injury and improved outcomes of secondary Pseudomonas aeruginosa infections following caecal ligation and puncture, thus potentially leading to a highly innovative and translational approach to treat sepsis efficiently and safely.

Multifunctional nanoparticle potentiates the in situ vaccination effect of radiation therapy and enhances response to immune checkpoint blockade.

Radiation therapy (RT) activates an in situ vaccine effect when combined with immune checkpoint blockade (ICB), yet this effect may be limited because RT does not fully optimize tumor antigen presentation or fully overcome suppressive mechanisms in the tumor-immune microenvironment. To overcome this, we develop a multifunctional nanoparticle composed of polylysine, iron oxide, and CpG (PIC) to increase tumor antigen presentation, increase the ratio of M1:M2 tumor-associated macrophages, and enhance stimulation of a type I interferon response in conjunction with RT. In syngeneic immunologically "cold" murine tumor models, the combination of RT, PIC, and ICB significantly improves tumor response and overall survival resulting in cure of many mice and consistent activation of tumor-specific immune memory. Combining RT with PIC to elicit a robust in situ vaccine effect presents a simple and readily translatable strategy to potentiate adaptive anti-tumor immunity and augment response to ICB or potentially other immunotherapies.

External stimuli-responsive nanoparticles for spatially and temporally controlled delivery of CRISPR-Cas genome editors.

The CRISPR-Cas9 system is a powerful tool for genome editing, which can potentially lead to new therapies for genetic diseases. To date, various viral and non-viral delivery systems have been developed for the delivery of CRISPR-Cas9 in vivo. However, spatially and temporally controlled genome editing is needed to enhance the specificity in organs/tissues and minimize the off-target effects of editing. In this review, we summarize the state-of-the-art non-viral vectors that exploit external stimuli (i.e., light, magnetic field, and ultrasound) for spatially and temporally controlled genome editing and their in vitro and in vivo applications.

Hydrogen peroxide-responsive platelet membrane-coated nanoparticles for thrombus therapy.

Occlusion of blood vessels caused by thrombi is the major pathogenesis of various catastrophic cardiovascular diseases. Thrombi can be prevented or treated by antithrombotic drugs. However, free antithrombotic drugs often have relatively low therapeutic efficacy due to a number of limitations such as short half-life, unexpected bleeding complications, low thrombus targeting capability, and negligible hydrogen peroxide (H2O2)-scavenging ability. Inspired by the abundance of H2O2 and the active thrombus-targeting property of platelets, a H2O2-responsive platelet membrane-cloaked argatroban-loaded polymeric nanoparticle (PNPArg) was developed for thrombus therapy. Poly(vanillyl alcohol-co-oxalate) (PVAX), a H2O2-degradable polymer, was synthesized to form an argatroban-loaded nanocore, which was further coated with platelet membrane. The PNPArg can effectively target the blood clots due to the thrombus-homing property of the cloaked platelet membrane, and subsequently exert combined H2O2-scavenging effect via the H2O2-degradable nanocarrier polymer and antithrombotic effect via argatroban, the released payload. The PNPArg effectively scavenged H2O2 and protected cells from H2O2-induced cellular injury in RAW 264.7 cells and HUVECs. The PNPArg rapidly targeted the thrombosed vessels and remarkably suppressed thrombus formation, and the levels of H2O2 and inflammatory cytokines in the ferric chloride-induced carotid arterial thrombosis mouse model. Safety assessment indicated good biocompatibility of the PNPArg. Taken together, the biomimetic PNPArg offers multiple functionalities including thrombus-targeting, antioxidation, and H2O2-stimulated antithrombotic action, thereby making it a promising therapeutic nanomedicine for thrombosis diseases.

In vivo targeted delivery of nucleic acids and CRISPR genome editors enabled by GSH-responsive silica nanoparticles.

The rapid development of gene therapy and genome editing techniques brings up an urgent need to develop safe and efficient nanoplatforms for nucleic acids and CRISPR genome editors. Herein we report a stimulus-responsive silica nanoparticle (SNP) capable of encapsulating biomacromolecules in their active forms with a high loading content and loading efficiency as well as a well-controlled nanoparticle size (~50 nm). A disulfide crosslinker was integrated into the silica network, endowing SNP with glutathione (GSH)-responsive cargo release capability when internalized by target cells. An imidazole-containing component was incorporated into the SNP to enhance the endosomal escape capability. The SNP can deliver various cargos, including nucleic acids (e.g., DNA and mRNA) and CRISPR genome editors (e.g., Cas9/sgRNA ribonucleoprotein (RNP), and RNP with donor DNA) with excellent efficiency and biocompatibility. The SNP surface can be PEGylated and functionalized with different targeting ligands. In vivo studies showed that subretinally injected SNP conjugated with all-trans-retinoic acid (ATRA) and intravenously injected SNP conjugated with GalNAc can effectively deliver mRNA and RNP to murine retinal pigment epithelium (RPE) cells and liver cells, respectively, leading to efficient genome editing. Overall, the SNP is a promising nanoplatform for various applications including gene therapy and genome editing.

Injectable Hydrogel Capable of In Situ Covalent Crosslinking for Permanent Embolization.

Vascular embolization provides an effective approach for the treatment of hemorrhage, aneurysms, and other vascular abnormalities. However, current embolic materials, such as metallic coils and liquid embolic agents, are limited by their inability to provide safe, consistent, and controlled embolization. Here, we report an injectable hydrogel that can remain at the injection site and subsequently undergo in situ covalent crosslinking, leading to the formation of a dual-crosslinking network (DCN) hydrogel for endovascular embolization. The DCN hydrogel is simple to prepare, easy to deploy via needles and catheters, and mechanically stable at the target injection site, thereby avoiding embolization of nontarget vessels. It possesses efficient hemostatic activity and good biocompatibility. The DCN hydrogel is also clearly visible under X-ray imaging, thereby allowing for targeted embolization. In vivo tests in a rabbit artery model demonstrates that the DCN hydrogel is effective in achieving immediate embolization of the target artery with long-term occlusion by inducing luminal fibrosis. Collectively, the DCN hydrogel provides a viable, biocompatible, and cost-effective alternative to existing embolic materials with clinical translation potential for endovascular embolization.

A Dual-Responsive Antibiotic-Loaded Nanoparticle Specifically Binds Pathogens and Overcomes Antimicrobial-Resistant Infections.

Antimicrobial resistant (AMR) infections are a growing threat to public health and there is a general lack of development in new antibiotics. Here, a dextran-coated stimuli-responsive nanoparticle (NP) that encapsulates the hydrophobic antibiotic, rifampicin, and specifically binds bacteria to overcome AMR infections is reported. The NP shows a strong affinity with a variety of pathogens in vitro and effectively accumulates in the bacterial infected tissues. The NP is activated by either low pH or high reactive oxygen species in the infectious microenvironment, and releases both cationic polymer and rifampicin that display synergistic activity against AMR pathogens. The NP carrier also enables the antibiotic to penetrate both bacterial biofilms and mammalian cells, thus allowing the elimination of biofilm and intracellular infections. The NP formulation demonstrates both safety and efficacy in two animal infection models against either Gram-negative or Gram-positive AMR pathogens.