Gene Reprogramming Armed Macrophage Membrane-Camouflaged Nanoplatform Enhances Bionic Targeted Drug Delivery to Solid Tumor for Synergistic Therapy.

Efficient drug delivery to solid tumors remains a challenge. HER2-positive (HER2+) tumors are an aggressive cancer subtype with a resistance to therapy, high risk of relapse, and poor prognosis. Although nanomedicine technology shows obvious advantages in tumor treatment, its potential clinical translation is still impeded by the unsatisfactory delivery and therapeutic efficacy. In this study, a gene reprogramming macrophage membrane-encapsulated drug-loading nanoplatform was developed for HER2+ cancer therapy based on the co-assembly of poly (lactic-co-glycolic acid) (PLGA) nanoparticles and engineered modified macrophage membranes. In this nanoplatform, near-infrared (NIR) fluorescent dye ICG or chemotherapeutic drug doxorubicin (DOX) was loaded into the PLGA cores, and an anti-HER2 affibody was stably expressed on the membrane of macrophages. In comparison to the nanoparticles with conventional macrophage membrane coating, the ICG/DOX@AMNP nanoparticles armed with anti-HER2 affibody showed excellent HER2-targeting ability both in vitro and in vivo. Small animal imaging studies confirmed the improved pharmacokinetics of drug delivery and specific distribution of the ICG/DOX@AMNPs in HER2+ tumors. Mechanistically, compared with DOX@NPs or DOX@MNPs nanoparticles, DOX@AMNPs exhibited synergistic inhibition of HER2+ cancer cells or mice tumor growth by inducing apoptosis and blocking the PI3K/AKT signaling pathway. Altogether, this study proposes a promising biomimetic nanoplatform for the efficient targeted delivery of chemotherapeutic agents to HER2+ tumors, demonstrating its great potential for solid tumor therapy.

Post-Remedial Oxygen Supply: A New Perspective on Photodynamic Therapy to Suppress Tumor Metastasis.

Photodynamic therapy (PDT) holds great promise in tumor therapy due to high safety, efficacy, and specificity. However, the risk of increased metastasis in hypoxic tumors after oxygen-dependent PDT remains underestimated. Here, we propose a post-PDT oxygen supply (POS) strategy to reduce the risk of metastasis. Herein, biocompatible and tumor-targeting Ce6@BSA and PFC@BSA nanoparticles were constructed for PDT and POS in a 4T1-orthotropic breast cancer model. PDT with Ce6@BSA nanoparticles increased tumor metastasis via the HIF-1α signaling pathway, whereas POS significantly reduced the PDT-triggered metastasis by blocking this pathway. Furthermore, POS, with clinical protocols and an FDA-approved photosensitizer (hypericin), and oxygen inhalation reduced PDT-induced metastasis. Our study findings indicate that PDT may increase the risk of tumor metastasis and that POS may solve this problem. POS can reduce the metastasis resulting not only from PDT but also from other oxygen-dependent treatments such as radiotherapy and sonodynamic therapy.

Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes.

BACKGROUND: RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences-a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5' end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3' end. RESULTS: Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues. CONCLUSIONS: The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening.

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes.

RNA plays an important role in life processes. Imaging of messenger RNAs (mRNAs) and micro-RNAs (miRNAs) not only allows us to learn the formation and transcription of mRNAs and the biogenesis of miRNAs involved in various life processes, but also helps in detecting cancer. High-performance RNA imaging probes greatly expand our view of life processes and enhance the cancer detection accuracy. In this review, we summarize the state-of-the-art high-performance RNA imaging probes, including exogenous probes that can image RNA sequences with special modification and endogeneous probes that can directly image endogenous RNAs without special treatment. For each probe, we review its structure and imaging principle in detail. Finally, we summarize the application of mRNA and miRNA imaging probes in studying life processes as well as in detecting cancer. By correlating the structures and principles of various probes with their practical uses, we compare different RNA imaging probes and offer guidance for better utilization of the current imaging probes and the future design of higher-performance RNA imaging probes.

Relationship between peptide structure and antimicrobial activity as studied by de novo designed peptides.

As fundamental components in innate immunity, antimicrobial peptides (AMPs) hold great potentials in the treatment of persistent infections involving slow-growing or dormant bacteria in which, selective inhibition of prokaryotic bacteria in the context of eukaryotic cells is not only an essential requirement, but also a critical challenge in the development of antimicrobial peptides. To identify the sequence and structural properties critical for antimicrobial activity, a series of peptides varying in sequence, length, hydrophobicity/charge ratio, and secondary structure, were designed and synthesized. Their antimicrobial activities were then tested using Escherichia coli and HEK293 cells, together with several index activities against model membrane, including liposome leakage, fusion, and aggregation. While no evident correlation between the antimicrobial activity and the property of the peptides was observed, common activities against model membrane were nevertheless identified for the active antimicrobial peptides: mediating efficient membrane leakage, negligible membrane fusion and liposome aggregation. Therefore, in addition to identifying one highly active antimicrobial peptide, our study further sheds light on the design principle for these molecules.

Aggregation, fusion, and leakage of liposomes induced by peptides.

Biological membranes are heterogeneous systems. Their functions are closely related to the lipid lateral segregation in the presence of membrane proteins. In this work, we designed two peptides, amphiphilic cationic peptides K3L8K3 and nonamphiphilic peptides K20, and studied their interactions with binary liposomes in different phases (Lα, Lß', and Lα/Lß'). As mimics of membrane proteins, both K3L8K3 and K20 can cause the liposomes to aggregate, fuse, or leak. These processes were closely related to the phases of liposomes. For the liposomes in Lα phase, heavy aggregation, fusion, and leakage were observed in the presence of either K20 or K3L8K3. For the liposomes in Lß' phase, neither K3L8K3 nor K20 can induce fusion or leakage. For the liposomes in Lα/Lß' phase, K3L8K3 caused the liposomes to aggregate, fuse, and leak, while K20 only led to aggregation. The kinetics of aggregation, fusion, and leakage in each phase were recorded, and they were related to the lipid demixing in the presence of the peptide. Our work not only gained insight into the effect of the lipid demixing on the interactions between peptide and membrane, but also helped in developing drug delivery vehicles with liposomes as the platform.

Liposomes physically coated with peptides: preparation and characterization.

Physically coating liposomes with peptides of desirable functions is an economic, versatile, and less time-consuming approach to prepare drug delivery vehicles. In this work, we designed three peptides-Ac-WWKKKGGNNN-NH2 (W2K3), Ac-WWRRRGGNNN-NH2(W2R3), Ac-WWGGGGGNNN-NH2(W2G3)-and studied their coating ability on negatively charged liposomes. It was found that the coating was mainly driven by the electrostatic interaction between the peptides' cationic side groups and the acidic lipids, which also mediated the "anchoring " of Trp residuals in the interfacial region of lipid bilayers. At the same conditions, the amount of the coated W2R3 was more than that of W2K3, but the stability of the liposome coated with W2R3 was deteriorated. This was caused by the delocalized charge of the guanidinium group of arginine. The coating of the peptide rendered the liposome pH-responsive behavior but did not prominently change the phase transition temperature. The liposome coated with peptides displayed appropriate pH/temperature dual responsive characteristics and was able to release the content in a controlled manner.

Filamentous-Actin-Mimicking Nanoplatform for Enhanced Cytosolic Protein Delivery.

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.

Genetically engineered macrophages as living cell drug carriers for targeted cancer therapy.

Precise targeting is a major prerequisite for effective cancer therapy because it ensures a sufficient therapeutic dosage in tumors while minimizing off-target side effects. Herein, we report a live-macrophage-based therapeutic system for high-efficiency tumor therapy. As a proof of concept, anti-human epidermal growth factor receptor-2 (HER2) affibodies were genetically engineered onto the extracellular membrane of macrophages (AE-Mφ), which further internalized doxorubicin (DOX)-loaded poly(lactic-co-glycolic acid) nanoparticles (NPs) to produce a macrophage-based therapeutic system armed with anti-HER2 affibodies. NPs(DOX)@AE-Mφ were able to target HER2+ cancer cells and specifically elicit affibody-mediated cell therapy. Most importantly, the superior HER2 + -targeting capability of NPs(DOX)@AE-Mφ greatly guaranteed high accumulation at the tumor site for improved chemotherapy, which acted synergistically with cell therapy to significantly enhance anti-tumor efficacy. This study suggests that NPs(DOX)@AE-Mφ could be utilized as an innovative 'living targeted drug' platform for combining both macrophage-mediated cell therapy and targeted chemotherapy for the individualized treatment of solid tumors.

Engineered Macrophages-Based uPA-Scavenger Load Gemcitabine to Prompt Robust Treating Cancer Metastasis.

The majority of cancer patients die of metastasis rather than primary tumors, and most patients may have already completed the cryptic metastatic process at the time of diagnosis, making them intractable for therapeutic intervention. The urokinase-type plasminogen activator (uPA) system is proved to drive cancer metastasis. However, current blocking agents such as uPA inhibitors or antibodies are far from satisfactory due to poor pharmacokinetics and especially have to face multiplex mechanisms of metastasis. Herein, an effective strategy is proposed to develop a uPA-scavenger macrophage (uPAR-MΦ), followed by loading chemotherapeutics with nanoparticles (GEM@PLGA) to confront cancer metastasis. Interestingly, significant elimination of uPA by uPAR-MΦ is demonstrated by transwell analysis on tumor cells in vitro and enzyme-linked immunosorbent assay detection in peripheral blood of mice with metastatic tumors, contributing to significant inhibition of migration of tumor cells and occurrence of metastatic tumor lesions in mice. Moreover, uPAR-MΦ loaded with GEM@PLGA shows a robust antimetastasis effect and significantly prolonged survival in 4T1-tumor-bearing mice models. This work provides a novel living drug platform for realizing a potent treatment strategy to patients suffering from cancer metastasis, which can be further expanded to handle other tumor metastasis markers mediating cancer metastasis.

Liposome-templated gold nanoparticles for precisely temperature-controlled photothermal therapy based on heat shock protein expression.

Mild temperature photothermal therapy is gaining more and more attention due to high safety, high specificity and moderate efficacy. However, the therapeutical outcome of mild photothermal therapy is limited due to the overexpression of heat shock proteins (HSPs). Therefore, the precise management of HSP expression is the key to improvement of mild temperature photothermal therapy. However, the correlation between HSP expression and photothermal temperature in vivo is still unclear. To precisely control the photothermal temperature by managing the HSP expression, we quantified the HSP expression at different photothermal temperatures after irradiation on liposome-templated gold nanoparticles, which have high photostability, high photothermal conversion efficiency and low temperature fluctuation (smaller than 1 â„ƒ). We found that the expression of HSP70 was least at 47 â„ƒ, which was the optimal temperature for HSP management. We chose to co-administrate HSP70 inhibitor during 47 â„ƒ photothermal therapy, leading to greatly enhanced tumor inhibition. Our precise temperature-controlled photothermal therapy based on HSP expression offers a new strategy for clinical tumor photothermal therapy.

Liposomal Glucose Oxidase for Enhanced Photothermal Therapy and Photodynamic Therapy against Breast Tumors.

Organic near-infrared fluorescent dye mediated photothermal therapy (PTT) and photodynamic therapy (PDT) suffer from heat shock response, since, heat shock proteins (HSPs) are overexpressed and can repair the proteins damaged by PTT and PDT. Starvation therapy by glucose oxide (GOx) can inhibit the heat shock response by limiting the energy supply. However, the delivery of sufficient and active GOx remains a challenge. To solve this problem, we utilize liposomes as drug carriers and prepare GOx loaded liposome (GOx@Lipo) with a high drug loading content (12.0%) and high enzymatic activity. The successful delivery of GOx shows excellent inhibition of HSPs and enhances PTT and PDT. Additionally, we apply the same liposome formulation to load near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbo cyanine iodide (DiR) and prepare DiR contained liposomes (DiR@Lipo) for PTT and PDT. The liposomal formulation substantially enhances the PTT and PDT properties of DiR as well as the cellular uptake and tumor accumulation. Finally, the combination therapy shows excellent tumor inhibition on 4T1 tumor-bearing mice. Interestingly, we also find that the starvation therapy can efficiently inhibit tumor metastasis, which is probably due to the immunogenic effect. Our work presents a biocompatible and effective carrier for the combination of starvation therapy and phototherapy, emphasizing the importance of auxiliary starvation therapy against tumor metastasis and offering important guidance for clinical PTT and PDT.

Pyroptosis in inflammatory diseases and cancer.

Pyroptosis is a lytic and inflammatory type of programmed cell death that is usually triggered by inflammasomes and executed by gasdermin proteins. The main characteristics of pyroptosis are cell swelling, membrane perforation, and the release of cell contents. In normal physiology, pyroptosis plays a critical role in host defense against pathogen infection. However, excessive pyroptosis may cause immoderate and continuous inflammatory responses that involves in the occurrence of inflammatory diseases. Attractively, as immunogenic cell death, pyroptosis can serve as a new strategy for cancer elimination by inducing pyroptotic cell death and activating intensely antitumor immunity. To make good use of this double-edged sword, the molecular mechanisms, and therapeutic implications of pyroptosis in related diseases need to be fully elucidated. In this review, we first systematically summarize the signaling pathways of pyroptosis and then present the available evidences indicating the role of pyroptosis in inflammatory diseases and cancer. Based on this, we focus on the recent progress in strategies that inhibit pyroptosis for treatment of inflammatory diseases, and those that induce pyroptosis for cancer therapy. Overall, this should shed light on future directions and provide novel ideas for using pyroptosis as a powerful tool to fight inflammatory diseases and cancer.

Insight into the spatial interaction of D-π-A bridge derived cyanines and nitroreductase for fluorescent cancer hypoxia detection.

Nitroreductase (NTR) detection in tumor is critical because NTR level is correlated with hypoxia degree and cancer prognosis. With the feature of high sensitivity and selectivity, fluorescence organic probes for NTR detection exhibited a promising future for tumor hypoxia detection. However, the discovery and design of such probes have been impeded due to the lack of the understanding of spatial match and mismatch of these probes with NTR. Here, we have developed two new nitrophenyl-functionalized trimethincyanine (Cy3) probes with para- or meta- positions of nitro-group in phenyl ring. Para-nitrophenyl substituted Cy3 (pNP-Cy3) exhibited a remarkable response to NTR (20-fold fluorescence enhancement) with good selectivity and sensitivity. Experimental and theoretical analysis verified that the substituent position of nitro group on phenyl ring of dyes altered the spatial arrangement of nitro-substituent group, thereby modulated the spatial match and mismatch between Cy3 dyes and binding domain of NTR, and consequently led to a different fluorescent turn-on response. In tumor-bearing mice model, hypoxia status of A549 xenografted tumor of mice was successfully delineated by using pNP-Cy3. These results may provide a clue for designing new cyanine-derived NTR probe to monitor NTR-overexpressed hypoxia cancer cells.

Bioorthogonally activatable cyanine dye with torsion-induced disaggregation for in vivo tumor imaging.

Advancement of bioorthogonal chemistry in molecular optical imaging lies in expanding the repertoire of fluorophores that can undergo fluorescence signal changes upon bioorthogonal ligation. However, most available bioorthogonally activatable fluorophores only emit shallow tissue-penetrating visible light via an intramolecular charge transfer mechanism. Herein, we report a serendipitous "torsion-induced disaggregation (TIDA)" phenomenon in the design of near-infrared (NIR) tetrazine (Tz)-based cyanine probe. The TIDA of the cyanine is triggered upon Tz-transcyclooctene ligation, converting its heptamethine chain from S-trans to S-cis conformation. Thus, after bioorthogonal reaction, the tendency of the resulting cyanine towards aggregation is reduced, leading to TIDA-induced fluorescence enhancement response. This Tz-cyanine probe sensitively delineates the tumor in living mice as early as 5 min post intravenous injection. As such, this work discovers a design mechanism for the construction of bioorthogonally activatable NIR fluorophores and opens up opportunities to further exploit bioorthogonal chemistry in in vivo imaging.

Recent progress in drug delivery and cancer theranostic built from metal-organic framework.

With the improvement of living standards, cancer has become a great challenge around the world during last decades, meanwhile, abundant nanomaterials have been developed as drug delivery system (DDS) or cancer theranostic agents (CTAs) with their outstanding properties. However, low multifunctional efficiency and time-consuming synthesis limit their further applications. Nowadays, green chemistry, in particular, the concept of atom economy, has defined new criteria for the simplicity and efficient production of biomaterials for nanomedicine, which not only owns the property of spatio-temporal precision imaging, but also possess the ability to treat cancer. Interestingly, metal-organic framework (MOF) is an excellent example to meet the requirements behind this concept and has great potential for next-generation nanomedicine. In this review, we summarize our recent researches and inspiring progresses in designing DDS and CTA built from MOF, aiming to show the simplicity, control, and versatility, and provide views on the development of MOF-based nanomedicine in the future.

Albumin-based fluorescence resonance energy transfer nanoprobes for multileveled tumor tissue imaging and dye release imaging.

Tumor tissue imaging and drug release imaging are both crucial for tumor imaging and image-guided drug delivery. It is urgent to develop a multileveled tumor imaging platform to realize the multiple imaging applications. In this work, we synthesized an albumin-based fluorescence resonance energy transfer (FRET) probe Cy5/7@HSA NPs containing two near-infrared cyanine dyes (CyBI5 and CyBI7) with high FRET efficiency (97 %). Excellent brightness and efficient FRET inside Cy5/7@HSA NPs enabled high-sensitive cell imaging and tumor imaging. This unique nanoprobe imaged 4T1 tumor-bearing mice with high sensitivity (TBR = 5.2) at 24 h post-injection and the dyes penetrated the tumor interior around 4 h post-injection. The release of dyes from nanoprobes was also tracked. This result shows the strong potential of this albumin-based FRET nanoprobe as multileveled tumor imaging platform for in vivo tumor imaging, drug delivery and image-guided surgery.

Recognition of single- and double-stranded oligonucleotides by bovine serum albumin via nonspecific interactions.

The complexes formed by bovine serum albumin (BSA) with single-stranded oligonucleotide (ss-oligo) or double-stranded oligonucleotide (ds-oligo) were investigated by laser light scattering, zeta potential analysis, and atomic force microscopy. It was found that BSA was able to recognize ss-oligo and ds-oligo upon forming complexes in HCOOH-HCOONa buffer at pH 3.0. When oligonucleotide was added dropwise to BSA, BSA formed a complex with ss-oligo but not with ds-oligo in the studied charge ratio. When BSA was added to oligonucleotides, BSA formed complexes with both ss-oligo and ds-oligo but via different paths: the BSA/ds-oligo underwent two processes, heavy precipitation followed by reentry, with increasing BSA/oligo charge ratio, whereas BSA/ss-oligo underwent only aggregation process, but with a charge reversal occurred at BSA/oligo charge ratio about 0.1. Moreover, the complex formed by BSA and ds-oligo showed a kinetics much slower than that of BSA and ss-oligo. We attributed the big difference upon complexation to the physical nature of oligonucleotides as well as the conformational change of BSA under severe conditions. The differentiation of ss-oligo from ds-oligo by BSA via nonspecific interactions gained insight in the recognition of DNA or RNA by specific protein (enzyme) under physiological conditions.

Calming the Cytokine Storm in Pneumonia by Biomimetic Nanoparticles.

Recently, the Wang group at Soochow University and the Gu group at the University of California, Los Angeles demonstrated the targeting ability of platelet-derived extracellular vesicles to deliver anti-inflammatory drug [5-(p-fluorophenyl)-2-ureido] thiophene-3-carboxamide (TPCA-1) to pneumonia for calming the local cytokine storm in acute lung injury.

Engineering Macrophages for Cancer Immunotherapy and Drug Delivery.

Macrophages play an important role in cancer development and metastasis. Proinflammatory M1 macrophages can phagocytose tumor cells, while anti-inflammatory M2 macrophages such as tumor-associated macrophages (TAMs) promote tumor growth and invasion. Modulating the tumor immune microenvironment through engineering macrophages is efficacious in tumor therapy. M1 macrophages target cancerous cells and, therefore, can be used as drug carriers for tumor therapy. Herein, the strategies to engineer macrophages for cancer immunotherapy, such as inhibition of macrophage recruitment, depletion of TAMs, reprograming of TAMs, and blocking of the CD47-SIRPα pathway, are discussed. Further, the recent advances in drug delivery using M1 macrophages, macrophage-derived exosomes, and macrophage-membrane-coated nanoparticles are elaborated. Overall, there is still significant room for development in macrophage-mediated immune modulation and macrophage-mediated drug delivery, which will further enhance current tumor therapies against various malignant solid tumors, including drug-resistant tumors and metastatic tumors.