Eliminating Intracellular MRSA via Mannosylated Lipid-Coated Calcium Phosphate Nanoparticles.

Methicillin-resistant Staphylococcus aureus (MRSA) within cells proves exceptionally challenging to eradicate using conventional antimicrobials, resulting in recurring infections and heightened resistance. Herein, we reported an innovative mannosylated lipid-coated photodynamic/photothermal calcium phosphate nanoparticle (MAN-LCaP@ICG) for eradicating intracellular MRSA. The MAN-LCaP functioned as the vehicle for drug delivery, exhibiting preferential uptake by macrophages and facilitating the transport of ICG to intracellular pathogens. The MAN units integrated into MAN-LCaP@ICG could promote binding with MAN residuals on macrophage cells, as evidenced by cellular uptake assays using fluorescence microscopy and flow cytometry. Following its targeted accumulation, MAN-LCaP@ICG could enter into the cytoplasm and efficiently eradicate intracellular MRSA by a combination of the lysosome escape capability of CaP and the photodynamic and photothermal therapeutic effects of ICG. Furthermore, MAN-LCaP@ICG could kill MRSA more effectively than LCaP@ICG without MAN units or free ICG in a mouse peritoneal infection model. Therefore, MAN-LCaP@ICG provided a promising direction for human clinical application in combating intracellular infections.

Synthesis and evaluation of antisense oligonucleotides prodrug with G-quadruplex assembly and lysosome escape capabilities for oncotherapy.

The applications of antisense oligonucleotides (ASOs) in rare or common diseases treatment have garnered great attention in recent years. Nevertheless, challenges associated with stability and bioavailability still persist, hampering the efficiency of ASOs. This work presents an ASO prodrug with parallel G-quadruplex assembly and lysosome escape capabilities for oncotherapy. Our findings revealed that the end-assembled quadruplex structure effectively shielded the ASO from enzymatic degradation. Meanwhile, the conjugation of maleimide within the quadruplex enhanced cellular uptake, potentially offering an alternative cell entry mechanism that circumvents lysosome involvement. Notably, an optimized molecule, Mal2-G4-ASO, exhibited remarkable therapeutic effects both in vitro and in vivo. This work presents a promising avenue for enhancing the activity of nucleic acid drugs in oncotherapy and potentially other disease contexts.

Inducing Autophagy and Blocking Autophagic Flux via a Virus-Mimicking Nanodrug for Cancer Therapy.

Maximizing the therapeutic capacity of drugs by allowing them to escape lysosomal degradation is a long-term challenge for nanodrug delivery. Japanese encephalitis virus (JEV) has evolved the ability to escape the endosomal region to avoid degradation of internal genetic material by lysosomes and further induce upregulation of cellular autophagy for the purpose of their mass reproduction. In this work, to exploit the lysosome escape and autophagy-inducing properties of JEV for cancer therapy, we constructed a virus-mimicking nanodrug consisting of anti-PDL1 antibody-decorated JEV-mimicking virosome encapsulated with a clinically available autophagy inhibitor, hydroxychloroquine (HCQ). Our study indicated that the nanodrug can upregulate the autophagy level and inhibit the autophagic flux, thereby inducing the apoptosis of tumor cells, and further activating the immune response, which can greatly improve the antitumor and tumor metastasis suppression effects and provide a potential therapeutic strategy for tumor treatment.

[Ginsenoside Rg_3 based liposomes target delivery of dihydroartemisinin and paclitaxel for treatment of triple-negative breast cancer].

Ginsenoside Rg_3, an active component of traditional Chinese medicine(TCM), was used as the substitute for cholesterol as the membrane material to prepare the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin and paclitaxel. The effect of the prepared drug-loading liposomes on triple-negative breast cancer in vitro was evaluated. Liposomes were prepared with the thin film hydration method, and the preparation process was optimized by single factor experiments. The physicochemical properties(e.g., particle size, Zeta potential, and stability) of the liposomes were characterized. The release behaviors of drugs in different media(pH 5.0 and pH 7.4) were evaluated. The antitumor activities of the liposomes were determined by CCK-8 on MDA-MB-231 and 4T1 cells. The cell scratch test was carried out to evaluate the effect of the liposomes on the migration of MDA-MB-231 and 4T1 cells. Further, the targeting ability of liposomes and the mechanism of lysosome escape were investigated. Finally, H9c2 cells were used to evaluate the potential cardiotoxicity of the preparation. The liposomes prepared were spheroid, with uniform particle size distribution, the ave-rage particle size of(107.81±0.01) nm, and the Zeta potential of(2.78±0.66) mV. The encapsulation efficiency of dihydroartemisinin and paclitaxel was 57.76%±1.38% and 99.66%±0.07%, respectively, and the total drug loading was 4.46%±0.71%. The accumulated release of dihydroartemisinin and paclitaxel from the liposomes at pH 5.0 was better than that at pH 7.4, and the liposomes could be stored at low temperature for seven days with good stability. Twenty-four hours after administration, the inhibition rates of the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin(70 µmol·L~(-1)) and paclitaxel on MDA-MB-231 and 4T1 cells were higher than those of the positive control(adriamycin) and free drugs(P<0.01). Compared with free drugs, liposomes inhibited the migration of MDA-MB-231 and 4T1 cells(P<0.05). Liposomes demonstrated active targeting and lysosome escape. In particular, liposomes showed lower toxicity to H9c2 cells than free drugs(P<0.05), which indicated that the preparation had the potential to reduce cardiotoxicity. The findings prove that ginsenoside Rg_3 characterized by the combination of drug and excipient is an ideal substitute for lipids in liposomes and promoted the development of innovative TCM drugs for treating cancer.

Strategy for Cytoplasmic Delivery Using Inorganic Particles.

Endosome escape is a key process for intracellular uptake of intact biomolecules and therapeutics, such as nucleic acids. Lysosome escape is a more common pathway during endocytosis, while some biomolecular, organic and inorganic materials are found to enhance the endosome escape, and several mechanisms have been proposed accordingly. Specifically, some inorganic nanomaterials show their unique mechanisms of action for enhanced endosome escape, including salt osmotic effect and gas blast effect. These inorganic nanomaterials are basically weakly alkaline and are naturally featured with the anti-acidification capacity, with limited solubility in neutral solutions. This review paper has briefly presented the strategies in the design of inorganic nanoparticle-based cellular delivery vehicles with endosome escapability and discussed a few typical inorganic nanomaterials that are currently widely examined for delivery purpose. A brief summary and prospect for this kind of inorganic nanomaterials are provided.

Dual pH/ROS-Responsive Nanoplatform with Deep Tumor Penetration and Self-Amplified Drug Release for Enhancing Tumor Chemotherapeutic Efficacy.

Poor deep tumor penetration and incomplete intracellular drug release remain challenges for antitumor nanomedicine application in clinical settings. Herein, a nanomedicine (RLPA-NPs) is developed that can achieve prolonged blood circulation, deep tumor penetration, active-targeting of cancer cells, endosome/lysosome escape, and intracellular selectivity self-amplified drug release for effective drug delivery. The RLPA-NPs are constructed by encapsulation of a pH-sensitive polymer octadecylamine-poly(aspartate-1-(3-aminopropyl) imidazole) (OA-P(Asp-API)) and a ROS-generation agent, ß-Lapachone (Lap), in micelles assembled by the tumor-penetration peptide internalizing RGD (iRGD)-modified ROS-responsive paclitaxel (PTX)-prodrug. iRGD could promote RLPA-NPs penetration into deep tumor tissue, and specific targeting to cancer cells. After internalization by cancer cells through receptor-mediated endocytosis, OA-P(Asp-API) can rapidly protonate in the endosome's acidic environment, resulting in RLPA-NPs escape from the endosome through the "proton sponge effect". At the same time, the RLPA-NPs micelle disassembles, releasing Lap and PTX-prodrug. Subsequently, the released Lap could generate ROS, consequently amplifying and accelerating PTX release to kill tumor cells. The in vitro and in vivo studies demonstrated that RLPA-NPs can significantly improve the therapeutic effect compared to control groups. Therefore, RLPA-NPs are a promising nanoplatform for overcoming multiple physiological and pathological barriers to enhance drug delivery.

Triton X-100-Modified Adenosine Triphosphate-Responsive siRNA Delivery Agent for Antitumor Therapy.

Modified polyethyleneimine (PEI) has been widely used as siRNA delivery agents. Here, a new Triton X-100-modified low-molecular-weight PEI siRNA delivery agent is developed together with the coupling of 4-carboxyphenylboronic acid (PBA) and dopamine grafted vitamin E (VEDA). Triton X-100, a nonionic detergent, greatly improves the cellular uptake of siRNA as well as the siRNA escape from endosome/lysosome because of its high transmembrane ability. In addition, the boronate bond between PBA and VEDA of the transfection agent can be triggered to release its entrapped siRNA because of the high level of adenosine triphosphate (ATP) in cancer cells. The transfection agent is successfully applied to deliver siRNAs targeting endogenous genes of epidermal growth factor receptor (EGFR) and kinesin-5 (Eg5) to cancer cells, showing good results on Eg5 and EGFR silencing ability and inhibition of cancer cell migration. Further in vivo study indicates that the Triton X-100-modified transfection agent is also efficient to deliver siRNA to cancer cells and shows significant tumor growth inhibition on mice tumor models. These results indicate that the Triton X-100-modified ATP-responsive transfection agent is a promising gene delivery vector for target gene silencing in vitro and in vivo.

Hexahistidine-Metal Assemblies: A Facile and Effective Codelivery System of Subunit Vaccines for Potent Humoral and Cellular Immune Responses.

Fully effective vaccines must induce both potent humoral and cellular immunities. Nanoparticles coencapsulating antigens and adjuvants have shown promising advantages as subunit vaccines in many aspects. However, the low loading efficiency and complicated synthesis process of these nanomaterials need to be improved. Here, we utilized hexahistidine (His6)-metal assembly (HmA) particles as carriers to codeliver ovalbumin peptides and cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs). We found that antigen/adjuvant-carrying HmA can efficiently enter into antigen-presenting cells and help the antigens escape from lysosomes to induce the maturation of these cells in vitro, characterized by increasing expression levels of costimulatory molecules and cytokines. More importantly, the vaccines with high biocompatibility can elicit strong humoral and cellular immunities by improving secretion of specific antibodies and cytokines, enhancing activation of DCs and T cells in vivo. Our results suggest that HmA provides a new approach for subunit vaccines by codelivery of antigens and adjuvants.

[Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure].

OBJECTIVE: To investigate the influence of the protamine sulfate on endocytosis and intracellular stability of tetrahedral framework nucleic acid (tFNA). METHODS: Articular cartilage cells were collected from 3-day-old C57BL mice. Cells at passage 1-2 were used in the experiments. 4 single-strand DNAs (S1 was marked by Cy5) were utilized to synthesize tFNAs via annealing process and ultrafiltration for purification. High-performance capillary electrophoresis (HPCE) was used to verify synthesis of tFNAs and transmission electron microscope was used to photo morphological characteristics. The 1 mg/mL protamine sulfate solution was slowly dropped into newly synthesized tFNAs (N/P=5/1). Then, Zeta potential was detected. Cells were treated with 100 nmol/L tFNAs with protamine sulfate in Dulbecco's Modified Eagle's medium (DMEM) (Exp.1), 100 nmol/L tFNAs in DMEM (Exp.2), and DMEM (Control), respectively. Flow cytometry was used to quantitatively detect intracellular Cy5 fluorescence after 6 h and 12 h treatments. Immunofluorescence staining was used to qualitatively observe internalized Cy5 fluorescence after 12 h treatment by laser confocal microscope. Lysosome of living cells were stained with lysosome probe. Colocalization between lysosome and tFNAs was observed by laser confocal microscope. RESULTS: After incubating protamine sulfate, negative potential was transformed into positive one ( (-1.567±0.163) mV to (4.700±0.484) mV). The fluorescence intensity of tFNAs in the Exp.1 group was higher than that of the Exp.2 group in 6 h and 12 h ( P<0.05). This was consistent with the results of immunofluorescence staining after 12 h. Colocalization of Cy5 fluorescence and lysosome in the Exp.1 group was more rare than that in the Exp.2 group at 6 h and 12 h. Furthermore, a large amount of Cy5 fluorescence was still seen in the Exp.1 group at 12 h, while Cy5 fluorescence of the Exp.2 group was less. CONCLUSION: Protamine sulfate can effectively enhance endocytosis, and to some extent it can achieve lysosome escape of tFNAs.

Light-Induced Self-Escape of Spherical Nucleic Acid from Endo/Lysosome for Efficient Non-Cationic Gene Delivery.

Developing non-cationic gene carriers and achieving efficient endo/lysosome escape of functional nucleic acids in cytosol are two major challenges faced by the field of gene delivery. Herein, we demonstrate the concept of self-escape spherical nucleic acid (SNA) to achieve light controlled non-cationic gene delivery with sufficient endo/lysosome escape capacity. In this system, Bcl-2 antisense oligonucleotides (OSAs) were conjugated onto the surface of aggregation-induced emission (AIE) photosensitizer (PS) nanoparticles to form core-shell SNA. Once the SNAs were taken up by tumor cells, and upon light irradiation, the accumulative 1 O2 produced by the AIE PSs ruptured the lysosome structure to promote OSA escape. Prominent in vitro and in vivo results revealed that the AIE-based core-shell SNA could downregulate the anti-apoptosis protein (Bcl-2) and induce tumor cell apoptosis without any transfection reagent.

Exploration of Antigen Induced CaCO3 Nanoparticles for Therapeutic Vaccine.

Therapeutic vaccines possess particular advantages and show promising potential to combat burdening diseases, such as acquired immunodeficiency syndrome, hepatitis, and even cancers. An efficient therapeutic vaccine would strengthen the immune system and eventually eliminate target cells through cytotoxic T lymphocytes (CTLs). Unfortunately, insufficient efficacy in triggering such an adaptive immune response is a problem that remains unsolved. To achieve efficient cellular immunity, antigen-presenting cells must capture and further cross-present disease-associated antigens to CD8 T cells via major histocompatibility complex I molecules. Here, a biomimetic strategy is developed to fabricate hierarchical ovalbumin@CaCO3 nanoparticles (OVA@NP, ≈500 nm) under the templating effect of antigen OVA. Taking advantage of the unique physicochemical properties of crystalline vaterite, cluster structure, and high loading, OVA@NP can efficiently ferry cargo antigen to dendritic cells and blast lysosomes for antigen escape to the cytoplasm. In addition, the first evidence that the physical stress from generated CO2 induces autophagy through the LC3/Beclin 1 pathways is presented. These outcomes cooperatively promote antigen cross-presentation, elicit CD8 T cell proliferation, ignite a potent and specific CTL response, and finally achieve prominent tumor therapy effects.

Tat/HA2 Peptides Conjugated AuNR@pNIPAAm as a Photosensitizer Carrier for Near Infrared Triggered Photodynamic Therapy.

To achieve an efficiency of intracellular photosensitizers (PSs) delivery and efficacy of photodynamic therapy, we have developed a novel class of PS formulation for encapsulating sulfonated aluminum phthalocyanine (AlPcS4) by taking advantage of the membrane-disruptive peptides Tat/HA2 and the photothermally triggered delivery system using AuNR@pNIPAAm. The coordinated effects of cell penetrating peptide Tat and fusogenic peptide HA2 could enhance the efficient cellular internalization and endo/lysosome escape of PSs delivery systems. Singlet oxygen generation was inhibited due to the reaction between loaded AlPcS4 and Au nanorods, which indicated that the AlPcS4-loaded, AuNR@pNIPAAm delivery system might be nonphototoxic in the circulatory system. However, this PSs-loaded nanosystem became highly phototoxic as it underwent the near-infrared irradiation by using the combined lights of 808 and 680 nm. Upon irradiation, the Tat/HA2 conjugated AuNR@pNIPAAm-Pc elicited an active photodynamic response against the cancer cells, leading to effective cells killing via mitochondria-associated apoptotic pathway. This study also demonstrated improved PDT therapeutic efficacy after intravenous administration of Tat/HA2-AuNR@pNIPAAm-Pc and the subsequent lights irradiations in tumor-bearing mice. We describe here a strategy for enhanced photodynamic eradication of solid tumors by endo/lysosomal escape and highlight the great promise of peptide-based nanocarriers used for cancer therapy.

Facile Construction of Chloroquine Containing PLGA-Based pDNA Delivery System for Efficient Tumor and Pancreatitis Targeting in Vitro and in Vivo.

Chloroquine diphosphate (CQ) was ingeniously used to take place of phosphate salt in traditional calcium phosphate coprecipitation method for pDNA transfection. With multiple roles of CQ in the novel Ca-CQ-pDNA complex including pDNA compaction and assistance in lysosome escape, the transfection efficiency of the pDNA was significantly increased relative to the traditional method. CQ did not intercalate into the DNA double helix as free CQ did, which was probably ascribed to the prior mixing of the pDNA with high concentration of calcium chloride. In order to construct efficacious vector for in vivo gene delivery, Ca-CQ-pDNA-PLGA-NPs was designed and prepared. With entrapment efficiency, particle size and pDNA integrity as screening conditions, the optimal prescription was obtained and CaPi-pDNA-PLGA-NPs made with classic calcium phosphate coprecipitation method after optimization was also prepared as control to systematically study the role of CQ in the novel vector. Physical characters of the vectors were comprehensively studied using TEM, DSC, and XRD. The safety of the vector both in vitro and in vivo was evaluated using MTT, hemolysis test, and histological sections. The Ca-CQ-pDNA-PLGA-NPs dramatically enhanced the gene tranfection efficiency in Human Embryonic kidney HEK293 cells compared with the CaPi-pDNA-PLGA-NPs and presented an increasing gene transfection for up 144 h. The relative fast release of the CQ compared with pDNA from the nanoparticles was responsive for the increased transfection. The Did-labeled-Ca-CQ-pDNA-PLGA-NPs exhibited excellent tumor targeting efficiency and sustained circulation time in CT26 mouse model. The Ca-CQ-pDNA-PLGA-NP loaded with the plasmid pVITRO2 expressing mSurvivin-T34A protein gave 70% tumor inhibition rate, which was partially ascribed to CQ. The Ca-CQ-pDNA-PLGA-NPs showed high targeting efficiency in C57 acute pancreatitis model. In all, the Ca-CQ-pDNA-PLGA-NP was a promising candidate for targeted gene delivery to both tumor and pancreatitis.

Induced Self-Assembly of Vitamin E-Spermine/siRNA Nanocomplexes via Spermine/Helix Groove-Specific Interaction for Efficient siRNA Delivery and Antitumor Therapy.

Gene therapy has been one of potential strategies for the treatment of different diseases, where efficient and safe gene delivery systems are also extremely in need. Current lipid nanoparticles (LNP) technology highly depends on the packing and condensation of nucleic acids with amine moieties. Here, an attempt to covalently link two natural compounds, spermine and vitamin E, is made to develop self-assembled nucleic acid delivery systems. Among them, the spermine moieties specifically interact with the major groove of siRNA helix through salt bridge interaction, while vitamin E moieties are located around siRNA duplex. Such amphiphilic vitamin E-spermine/siRNA complexes can further self-assemble into nanocomplexes like multiblade wheels. Further studies indicate that these siRNA nanocomplexes with the neutrally charged surface of vitamin E can enter cells via caveolin/lipid raft mediated endocytosis pathway and bypass lysosome trapping. With these self-assembled delivery systems, efficient siRNA delivery is successfully achieved for Eg5 and Survivin gene silencing as well as DNA plasmid delivery. Further in vivo study indicates that VE-Su-Sper/DSPE-PEG2000/siSurvivin self-assembled nanocomplexes can accumulate in cancer cells and gradually release siRNA in tumor tissues and show significant antitumor effect in vivo. The self-assembled delivery system provides a novel strategy for highly efficient siRNA delivery.

Ligand Phase Separation-Promoted, "Squeezing-Out" Mode Explaining the Mechanism and Implications of Neutral Nanoparticles That Escaped from Lysosomes.

Neutral nanomaterials functionalized with PEG or similar molecules have been popularly employed as nanomedicines. Compared to positive counterparts that are capable of harnessing the well-known proton sponge effect to facilitate their escape from lysosomes, it is yet unclear how neutral substances got their entry into the cytosol. In this study, by taking PEGylated, neutral Au nanospheres as an example, we systematically investigated their time-dependent translocation postuptake. Specifically, we harnessed dissipative particle dynamics simulations to uncover how nanospheres bypass lysosomal entrapment, wherein a mechanism termed as "squeezing-out" mode was discovered. We next conducted a comprehensive investigation on how nanomaterials implicate lysosomes in terms of integrity and functionality. By using single-molecule imaging, specific preservation of PEG-terminated with targeting moieties in lysosomes supports the "squeezing-out" mode as the mechanism underlying the lysosomal escape of nanomaterials. All evidence points out that such a process is benign to lysosomes, wherein the escape of nanomaterials proceeds at the expense of targeting moieties loss. Furthermore, we proved that by fine-tuning of the efficacy of nanomaterials escaping from lysosomes, modulation of distinct pathways and metabolic machinery can be achieved readily, thereby offering us a simple and robust tool to implicate cells.

NIR-Actuated Ferroptosis Nanomotor for Enhanced Tumor Penetration and Therapy.

Ferroptosis nano-inducers have drawn considerable attention in the treatment of malignant tumors. However, low intratumoral hydrogen peroxide level and complex biological barriers hinder the ability of nanomedicines to generate sufficient reactive oxygen species (ROS) and achieve tumor penetration. Here a near-infrared (NIR)-driven ROS self-supplying nanomotor is successfully designed for synergistic tumor chemodynamic therapy (CDT) and photothermal therapy (PTT). Janus nanomotor is created by the asymmetrical modification of polydopamine (PDA) with zinc peroxide (ZnO2) and subsequent ferrous ion (Fe2+) chelation via the polyphenol groups from the PDA, here refer as ZnO2@PDA-Fe (Z@P-F). ZnO2 is capable of slowly releasing hydrogen peroxide (H2O2) into an acidic tumor microenvironment (TME) providing sufficient ingredients for the Fenton reaction necessary for ferroptosis. Upon NIR laser irradiation, the loaded Fe2+ is released and a thermal gradient is simultaneously formed owing to the asymmetric PDA coating, thus endowing the nanomotor with self-thermophoresis based enhanced diffusion for subsequent lysosomal escape and tumor penetration. Therefore, the release of ferrous ions (Fe2+), self-supplied H2O2, and self-thermophoresis of nanomotors with NIR actuation further improve the synergistic CDT/PTT efficacy, showing great potential for active tumor therapy.

Fluorinated Lipid Nanoparticles for Enhancing mRNA Delivery Efficiency.

Lipid nanoparticles (LNPs), a nonviral nucleic acid delivery system, have shown vast potential for vaccine development and disease treatment. LNPs assist mRNA to cross physiological barriers such as cell membranes and endosomes/lysosomes, promoting the intracellular presentation of mRNA. However, the endosome escape efficiency and biosafety of currently commercialized LNPs are still unsatisfactory, resulting in underutilization of mRNA. Herein, we report that fluorinated modification of the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (PEG-DSPE), termed as FPD, in the LNPs can improve the delivery efficiency of mRNA. FPD accounts for only 1.5% of lipids in LNPs but could mediate a 5-fold and nearly 2-fold enhancement of mRNA expression efficiency in B16F10 tumor cells and primary dendritic cells, respectively. Mechanism studies reveal that FPD promotes the cellular internalization of LNPs as well as endosome escape. In vivo studies substantiate that FPD can augment overall mRNA expression at least 3-fold, either by intravenous or intraperitoneal injection, compared to LNPs prepared with nonfluorinated PEG-lipids at a relatively low mRNA dose. Besides, with the introduction of FPD, mRNA expression in the spleen augmented compared to that of the DMG-PEG commercial formulations. Benefiting from a prudent dosage of fluorine, the fluorinated LNPs display favorable biosafety profiles at cellular and zoological levels.

Electrostatic Attractive Self-Delivery of siRNA and Light-Induced Self-Escape for Synergistic Gene Therapy.

Small interfering RNA (siRNA) holds immense promise for suppressing gene expression and treating various life-threatening diseases, including cancer. However, efficient delivery and lysosomal escape remain critical challenges that hinder the therapeutic effectiveness of siRNA. Herein, cationic photosensitizer (NB-Br) is grafted onto polo-like kinase 1 (PLK1) siRNA to form an amphiphilic siRNA-photosensitizer conjugate (siPLK1-NB), which can self-assemble into nanoparticles (siPLK1-NB NPs) via electrostatic attraction. Notably, siPLK1-NB NPs exhibit rapid and efficient cell endocytosis, as well as outstanding tumor-targeting property in multiple tumor-bearing mice models. When siPLK1-NB NPs are located inside tumor cell lysosomes, the generated reactive oxygen species (ROS) after photoactivation can disrupt the lysosome membrane structure and facilitate siRNA escape from lysosomes. Under light irradiation, siPLK1-NB NPs can downregulate PLK1 expression and induce photodynamic killing, effectively inhibiting tumor cell growth both in vitro and in vivo. Consequently, this study provides a novel design strategy for carrier-free siRNA delivery systems. As far as it is known, this is the first report of a carrier-free siRNA delivery system based on electrostatic attraction.

Dual factor coactivatable fluorescent nanosensor with boosted cytoplasmic biomarker accessibility toward selective tumor imaging.

Fluorescent nanosensor-based tumor imaging holds great promise in cancer diagnosis and treatment assistance, yet the signal contrast is heavily hampered by the unspecific/unwanted activation at microscopic regions with a highly restricted local abundance of biomarkers. Herein, we developed an activation boosting strategy by the integration and manipulation of dual-factor coactivation of sensing and lysosome escape facilitated the rise of cytosolic biomarker accessibility. By employing hybrid DNA probes on gold nanoquenchers, ATP sensing initiated conformation switch of the corresponding aptamer units triggered the exposure of a hidden toehold in a loop structure. Sequentially, miRNA-21 sensing was triggered by toehold-mediated strand displacement and detachment of the binding complexes. The application of lysosomotropic agent chloroquine at optimized time interval facilitated the release of nanosensors into the cytosol and a ∼10.5-fold increment of intracellular fluorescence in vitro, while coactivation improved the cancer-to-normal cell signal ratio by ∼5.9 times. The synergy effects led to a high tumor-to-normal tissue ratio value of ∼7.9 in the in vivo imaging results. This strategy establishes a new paradigm of fluorescent nanosensors for selective and specific tumor imaging.

Black Phosphorus Nanosheets Assist Nanoerythrosomes for Efficient mRNA Vaccine Delivery and Immune Activation.

Messenger RNA (mRNA)-based vaccines have enormous potential in infectious disease prevention and tumor neoantigen application. However, developing an advanced delivery system for efficient mRNA delivery and intracellular release for protein translation remains a challenge. Herein, a biocompatible biomimetic system is designed using red blood cell-derived nanoerythrosomes (NER) and black phosphorus nanosheets (BP) for mRNA delivery. BP is covalently modified with polyethyleneimine (PEI), serving as a core to efficiently condense mRNA via electrostatic interactions. To facilitate the spleen targeting of the mRNA-loaded BP (BPmRNA ), NER is co-extruded with BPmRNA to construct a stable "core-shell" nanovaccine (NER@BPmRNA ). The mRNA nanovaccine exhibits efficient protein expression and immune activation via BP-mediated adjuvant effect and enhanced lysosomal escape. In vivo evaluation demonstrates that the system delivery of mRNA encoding coronavirus receptor-binding domain (RBD) significantly increases the antibody titer and pseudovirus neutralization effect compared with that of NER without BP assistance. Furthermore, the mRNA extracted from mouse melanoma tissues is utilized to simulate tumor neoantigen delivered by NER@BPmRNA . In the vaccinated mice, BP-assisted NER for the delivery of melanoma mRNA can induce more antibodies that specifically recognize tumor antigens. Thus, BP-assisted NER can serve as a safe and effective delivery vehicle in mRNA-based therapy.