Engineered CpG-Loaded Nanorobots Drive Autophagy-Mediated Immunity for TLR9-Positive Cancer Therapy.

Smart nanorobots have emerged as novel drug delivery platforms in nanomedicine, potentially improving anti-cancer efficacy and reducing side effects. In this study, an intelligent tumor microenvironment-responsive nanorobot is developed that effectively delivers CpG payloads to Toll-like receptor 9 (TLR9)-positive tumors to induce autophagy-mediated cell death for immunotherapy. The nanorobots are fabricated by co-self-assembly of two amphiphilic triblock polymer peptides: one containing the matrix metallopeptidase 2 (MMP2)-cleaved GPLGVRGS motif to control the mechanical opening of the nanorobots and provide targeting capability for TLR-9-positive tumors and the other consisting of an arginine-rich GRRRDRGRS sequence that can condense nuclear acid payloads through electrostatic interactions. Using multiple tumor-bearing mouse models, it is investigated whether the intravenous injection of CpG-loaded nanorobots could effectively deliver CpG payloads to TLR-9-positive tumors and elicit anti-tumor immunity through TLR9 signaling and autophagy. Therefore, besides being a commonly used adjuvant for tumor vaccination, CpG-loaded nanorobots can effectively reprogram the tumor immunosuppressive microenvironment and suppress tumor growth and recurrence. This nanorobot-based CpG immunotherapy can be considered a feasible approach to induce anti-tumor immunity, showing great therapeutic potential for the future treatment of TLR9-positive cancers.

The clinical progress and challenges of mRNA vaccines.

Owing to the breakthroughs in the prevention and control of the COVID-19 pandemic, messenger RNA (mRNA)-based vaccines have emerged as promising alternatives to conventional vaccine approaches for infectious disease prevention and anticancer treatments. Advantages of mRNA vaccines include flexibility in designing and manipulating antigens of interest, scalability in rapid response to new variants, ability to induce both humoral and cell-mediated immune responses, and ease of industrialization. This review article presents the latest advances and innovations in mRNA-based vaccines and their clinical translations in the prevention and treatment of infectious diseases or cancers. We also highlight various nanoparticle delivery platforms that contribute to their success in clinical translation. Current challenges related to mRNA immunogenicity, stability, and in vivo delivery and the strategies for addressing them are also discussed. Finally, we provide our perspectives on future considerations and opportunities for applying mRNA vaccines to fight against major infectious diseases and cancers. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Biology-Inspired Nanomaterials > Lipid-Based Structures.

Reactivation of the tumor suppressor PTEN by mRNA nanoparticles enhances antitumor immunity in preclinical models.

Increasing clinical evidence has demonstrated that the deletion or mutation of tumor suppressor genes such as the gene-encoding phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in cancer cells may correlate with an immunosuppressive tumor microenvironment (TME) and poor response or resistance to immune checkpoint blockade (ICB) therapy. It is largely unknown whether the restoration of functional PTEN may modulate the TME and improve the tumor's sensitivity to ICB therapy. Here, we demonstrate that mRNA delivery by polymeric nanoparticles can effectively induce expression of PTEN in Pten-mutated melanoma cells and Pten-null prostate cancer cells, which in turn induces autophagy and triggers cell death-associated immune activation via release of damage-associated molecular patterns. In vivo results illustrated that PTEN mRNA nanoparticles can reverse the immunosuppressive TME by promoting CD8+ T cell infiltration of the tumor tissue, enhancing the expression of proinflammatory cytokines, such as interleukin-12, tumor necrosis factor-α, and interferon-γ, and reducing regulatory T cells and myeloid-derived suppressor cells. The combination of PTEN mRNA nanoparticles with an immune checkpoint inhibitor, anti-programmed death-1 antibody, results in a highly potent antitumor effect in a subcutaneous model of Pten-mutated melanoma and an orthotopic model of Pten-null prostate cancer. Moreover, the combinatorial treatment elicits immunological memory in the Pten-null prostate cancer model.

RNA Nanotechnology-Mediated Cancer Immunotherapy.

RNA molecules (e.g., siRNA, microRNA, and mRNA) have shown tremendous potential for immunomodulation and cancer immunotherapy. They can activate both innate and adaptive immune system responses by silencing or upregulating immune-relevant genes. In addition, mRNA-based vaccines have recently been actively pursued and tested in cancer patients, as a form of treatment. Meanwhile, various nanomaterials have been developed to enhance RNA delivery to the tumor and immune cells. In this review article, we summarize recent advances in the development of RNA-based therapeutics and their applications in cancer immunotherapy. We also highlight the variety of nanoparticle platforms that have been used for RNA delivery to elicit anti-tumor immune responses. Finally, we provide our perspectives of potential challenges and opportunities of RNA-based nanotherapeutics in clinical translation towards cancer immunotherapy.

A tumour-selective cascade activatable self-detained system for drug delivery and cancer imaging.

Achieving the activation of drugs within cellular systems may provide targeted therapies. Here we construct a tumour-selective cascade activatable self-detained system (TCASS) and incorporate imaging probes and therapeutics. We show in different mouse models that the TCASS system accumulates in solid tumours. The molecules show enhanced accumulation in tumour regions via the effect of recognition induced self-assembly. Analysis of the molecular penetration in tumour tissue shows that in vivo self-assembly increases the penetration capability compared to typical soft or hard nanomaterials. Importantly, the in vivo self-assembled molecules exhibit a comparable clearance pathway to that of small molecules, which are excreted from organs of the reticuloendothelial system (liver and kidney), while are relatively slowly eliminated from tumour tissues. Finally, this system, combined with the NIR probe, shows high specificity and sensitivity for detecting bladder cancer in isolated intact patient bladders.

Discovery of Inhibitors of Aurora/PLK Targets as Anticancer Agents.

Aurora and polo-like kinases control the G2/M phase in cell mitosis, which are both considered as crucial targets for cancer cell proliferations. Here, naphthalene-based Aurora/PLK coinhibitors as leading compounds were designed through in silico approach, and a total of 36 derivatives were synthesized. One candidate (AAPK-25) was selected under in vitro cell based high throughput screening with an IC50 value = 0.4 µM to human colon cancer cell HCT-116. A kinome scan assay showed that AAPK-25 was remarkably selective to both Aurora and PLK families. The relevant genome pathways were also depicted by microarray based gene expression analysis. Furthermore, validated from a set of in vitro and in vivo studies, AAPK-25 significantly inhibited the development of the colon cancer growth and prolonged the median survival time at the end of the administration (p < 0.05). To sum up, AAPK-25 has a great potential to be developed for a chemotherapeutic agent in clinical use.

In Situ Manipulation of Dendritic Cells by an Autophagy-Regulative Nanoactivator Enables Effective Cancer Immunotherapy.

Cellular immunotherapeutics aim to employ immune cells as anticancer agents. Ex vivo engineering of dendritic cells (DCs), the initial role of an immune response, benefits tumor elimination by boosting specific antitumor responses. However, directly activating DCs in vivo is less efficient and therefore quite challenging. Here, we designed a nanoactivator that manufactures DCs through autophagy upregulating in vivo directly, which lead to a high-efficiency antigen presention of DCs and antigen-specific T cells generation. The nanoactivator significantly enhances tumor antigen cross-presentation and subsequent T cell priming. Consequently, in vivo experiments show that the nanoactivators successfully reduce tumor growth and prolong murine survival. Taken together, these results indicate in situ DCs manipulation by autophagy induction is a promising strategy for antigen presentation enhancement and tumor elimination.

Peptide-Based Autophagic Gene and Cisplatin Co-delivery Systems Enable Improved Chemotherapy Resistance.

Cisplatin-based chemotherapy is a widely used first-line strategy for numerous cancers. However, drug resistances are often inevitable accompanied by the long-term use of cisplatin in vivo, significantly hampering its therapeutic efficacy and clinical outcomes. Among others, autophagy induction is one of the most common causes of tumor resistance to cisplatin. Herein, a self-assembled nanoprodrug platform was developed with the synergistic effect of cisplatin and RNAi to fight against cisplatin-resistant lung cancer. The nanoprodrug platform consists of three molecular modules, including prodrug complex of Pt(IV)-peptide-bis(pyrene), DSPE-PEG, and cRGD-modified DSPE-PEG. The Pt(IV) is immobilized with peptide via amide bonds, allowing the Pt(IV) to be loaded with a loading efficiency of >95% and rapid-release active platinum ions (Pt(II)) in the presence of glutathione (GSH). Meanwhile, the peptide of the prodrug complex could efficiently deliver Beclin1 siRNA ( Beclin1 is an autophagy initiation factor) to the cytoplasm, thereby leading to autophagy inhibition. In addition, incorporation of DSPE-PEG and cRGD-modified DSPE-PEG molecules improves the biocompatibility and cellular uptake of the nanoprodrug platform. In vivo results also indicate that the nanoprodrug platform significantly inhibits the growth of a cisplatin-resistant tumor on xenograft mice models with a remarkable inhibition rate, up to 84% after intravenous injection.

Progress in Tumor-Associated Macrophages: From Bench to Bedside.

Tumor-associated macrophages (TAMs) are of great interest in cancer immunology as they play an important role in the tumor microenvironment as cancer stromal cells recruited from circulating monocytes. TAMs are closely associated with tumor progression, including initiation, trophic growth, metabolism, angiogenesis, and metastasis; moreover, in clinical practice, their quantity can be related to poor prognosis. Fundamental and translational studies imply that TAMs are one of the most promising targets in tumor therapy. Herein, the biological origination and classification of TAMs, which correspond to their functions and differentiations, are reviewed in detail. In addition, recent basic research and clinical preprocess of TAMs in tumor immunotherapy are also discussed. Finally, the advances in the use of nanotechnology and TAMs for tumor therapy are discussed. This review focuses on the background and status of basic research and clinical significance of TAMs, points out the potential of TAMs in tumor immunological therapy, and clarifies the possibility of translation TAM-targeting therapies in medicine.

A self-destructive nanosweeper that captures and clears amyloid ß-peptides.

Cerebral amyloid ß-peptide (Aß) accumulation resulting from an imbalance between Aß production and clearance is one of the most important causes in the formation of Alzheimer's disease (AD). In order to preserve the maintenance of Aß homeostasis and have a notable AD therapy, achieving a method to clear up Aß plaques becomes an emerging task. Herein, we describe a self-destructive nanosweeper based on multifunctional peptide-polymers that is capable of capturing and clearing Aß for the effective treatment of AD. The nanosweeper recognize and bind Aß via co-assembly through hydrogen bonding interactions. The Aß-loaded nanosweeper enters cells and upregulates autophagy thus promoting the degradation of Aß. As a result, the nanosweeper decreases the cytotoxicity of Aß and rescues memory deficits of AD transgenic mice. We believe that this resourceful and synergistic approach has valuable potential as an AD treatment strategy.

Nanoantagonists with nanophase-segregated surfaces for improved cancer immunotherapy.

The blockade of PD-1/PD-L1 interaction by peptide antagonists can unleash and enhance pre-existing anti-cancer immune responses of T cells to eradicate cancer cells. However, low proteolytic stability is the "Achilles' Heel" of peptides. Here, we first report a nanoantagonist with a physiological temperature sensitive nanophase-segregated surface that exhibits significantly enhanced blood circulation, peptide stability and PD-L1 immune checkpoint blockade efficacy. Thermosensitive polymers with different phase transition temperatures (Tt) are used to form the nanophase-segregated surface on an Au nanorod core. Importantly, the nanophase-segregated surface aids the nanoantagonist to resist protein adsorption and enhance the systemic stability of the linked peptides. Finally, the as-designed nanoantagonist effectively blocks PD-1/PD-L1 interaction in vitro and in vivo, enhances the pre-existing CD8+ T cell tumor destruction capability and inhibits tumor growth. This study offers a new strategy for designing nano-formulations for cancer immunotherapy.

"In vivo self-assembled" nanoprobes for optimizing autophagy-mediated chemotherapy.

Autophagic therapy is regarded as a promising strategy for disease treatment. Appropriate autophagy regulations in vivo play a crucial role in translating this new concept from benchside to bedside. So far, emerging technologies are required to spatially and quantitatively monitor autophagic process in vivo in order to minimize the cytotoxity concerns associated with autophagy-mediated therapy. We successfully demonstrate the "proof-of-concept" study on autophagy-mediated chemotherapy in mice. Here, we describe a photoacoustic (PA) nanoprobe based on "in vivo self-assembly" idea for real-time and quantitative detection of autophagy in mice for the first time. The purpurin-18 (P18) monomer is connected to hydrophilic poly(amidoamine) dendrimer (4th generation) through a peptide (GKGSFGFTG) that can be cleaved by an autophagy-specific enzyme, i.e., ATG4B, consequently resulting in aggregation of P18 and enhanced PA signals. Based on this aggregation-induced "turn-on" PA signals, we noninvasively determine the ATG4B activity for monitoring autophagy of tumor in vivo. According to the results of PA imaging, we could optimize chemotherapy efficacy through precisely modulating autophagy, which thereby decrease systemic toxicity from chemotherapeutics and autophagy inhibitors. We envision it will pave the way for developing autophagy-based treatment of diseases in the future.

Photothermal Ring Integrated Intraocular Lens for High-Efficient Eye Disease Treatment.

Posterior capsule opacification (PCO) is the most common complication after cataract surgery. So far, the only method for PCO treatment is the precisely focused laser surgery. However, it causes severe complications such as physical damages and neuron impairments. Here, a nanostructured photothermal ring integrated intraocular lens (Nano-IOLs) is reported, in which the rim of commercially available IOLs (C-IOLs) is decorated with silica coated Au nanorods (Au@SiO2 ), for high-efficient prevention of PCO after cataract surgery. The Nano-IOLs is capable of eliminating the residual lens epithelial cells (LECs) around Nano-IOLs under mild laser treatment and block the formation of disordered LECs fibrosis, which eventually leads to the loss of vision. The Nano-IOLs shows good biocompatibility as well as extraordinary region-confined photothermal effect. In vivo studies reveal that PCO occurrence in rabbit models is about 30%-40% by using Nano-IOLs, which is significantly lower than the control group that treated with C-IOLs (100% PCO occurrence) 30 d postsurgery. To the best of our knowledge, it is the first example to integrate nanotechnology with intraocular implants aiming to clinically relevant PCO. Our findings indicate that spatial controllability of photothermal effect from nanomaterials may provide a unique way to intervene the PCO-induced loss of vision.

Recent Advances in Nanotechnology for Autophagy Detection.

Autophagy is closely related to various diseases, and is a diagnostic and therapeutic target for some diseases. In recent years, tremendous efforts have been made to develop excellent probes for detection of autophagy. Nanostructure-based probes are interesting and promising approaches for in vivo biological imaging due to their unique structural and functional characteristics, e.g., modulating pharmacokinetics property by biocompatible coatings, multimodality capacity by delivering multiple imaging agents and highly specific targeting by antibody ligands. In this Review, we first introduce recent advancements in the development of nanostructure-based probes for detection of autophagy, including inorganic hybrid nanomaterials and self-assembled peptide polymeric nanoparticles. Meanwhile, a nanoprobe based on a "in vivo self-assembly" strategy is highlighted. The "in vivo self-assembly" endows nanoprobes with higher accumulation, and longer and better signal stability for in vivo detection of autophagy. Furthermore, this novel strategy could be widely used for biomedical imaging/diagnostics and therapeutics, which would attract more attention to this research area.

General Approach of Stimuli-Induced Aggregation for Monitoring Tumor Therapy.

Intracellular construction of nanoaggregates from synthetic molecules to mimic natural ordered superstructures has gained increasing attention recently. Here, we develop an endogenous stimuli-induced aggregation (eSIA) approach to construct functional nanoaggregates for sensing and monitoring cellular physiological processes in situ. We design a series of thermosensitive polymer-peptide conjugates (PPCs), which are capable of constructing nanoaggregates in cells based on their isothermal phase transition property. The PPCs are composed of three moieties (i.e., a thermoresponsive polymer backbone, a grafted peptide, and a signal-molecule label). The bioenvironment-associated phase transition behavior of PPCs are carefully studied by consideration of various crucial parameters such as chain length, hydrophilicity, ratio of grafted peptides, and concentration. Intriguingly, under the specific intracellular stimulus, the PPCs are tailored and simultaneously form nanoaggregates exhibiting long-term retention effect, which enables specific identification and quantification of endogenous factors. This general approach is expected for high-performance in situ sensing and dynamic monitoring of disease progression in living subjects.

Polymer-KLAK Peptide Conjugates Induce Cancer Cell Death through Synergistic Effects of Mitochondria Damage and Autophagy Blockage.

Nanoscaled polymer-peptide conjugates (PPCs) containing both functional peptides and synthetic polymer comprise a new family of biomaterials that can circumvent the limitation of peptides alone. Our previous work showed that PPCs with the therapeutic peptide KLAK, especially PPCs with shorter PEG spacers and a higher degree of polymerization, exhibit enhanced antitumor effects through disrupting mitochondrial membranes. However, as PPCs have a spherical nanostructure (45-60 nm), this may have other effects besides the conjugated therapeutic peptide KLAK itself when they enter cancer cells. In this research, we compared the proteome differences of U87 cells treated with KLAK, polymer, and their conjugates (P-KLAK) through quantitative proteomics technology. The result reveals that proteins involved in oxidative stress response and the Nrf2/ARE pathway were significantly up-regulated after P-KLAK treatment. Moreover, the overexpression of sequestosome 1, a protein substrate that is selectively incorporated into the formation of autophagosome and degraded by autophagy, is found in our study and has not been reported previously in the study of KLAK toxicity. Additional experiments suggest that upon endocytosis, P-KLAK causes lysosome impairment and results in autophagosomes accumulation. Hence, P-KLAK might induce U87 cell death by autophagy blockage due to lysosome impairment as well as mitochondria damage synergistically.

Transformable Nanomaterials as an Artificial Extracellular Matrix for Inhibiting Tumor Invasion and Metastasis.

Tumor metastasis is one of the big challenges in cancer treatment and is often associated with high patient mortality. Until now, there is an agreement that tumor invasion and metastasis are related to degradation of extracellular matrix (ECM) by enzymes. Inspired by the formation of natural ECM and the in situ self-assembly strategy developed in our group, herein, we in situ constructed an artificial extracellular matrix (AECM) based on transformable Laminin (LN)-mimic peptide 1 (BP-KLVFFK-GGDGR-YIGSR) for inhibition of tumor invasion and metastasis. The peptide 1 was composed of three modules including (i) the hydrophobic bis-pyrene (BP) unit for forming and tracing nanoparticles; (ii) the KLVFF peptide motif that was inclined to form and stabilize fibrous structures through intermolecular hydrogen bonds; and (iii) the Y-type RGD-YIGSR motif, derived from LN conserved sequence, served as ligands to bind cancer cell surfaces. The peptide 1 formed nanoparticles (1-NPs) by the rapid precipitation method, owing to strong hydrophobic interactions of BP. Upon intravenous injection, 1-NPs effectively accumulated in the tumor site due to the enhanced permeability and retention (EPR) effect and/or targeting capability of RGD-YIGSR. The accumulated 1-NPs simultaneously transformed into nanofibers (1-NFs) around the solid tumor and further entwined to form AECM upon binding to receptors on the tumor cell surfaces. The AECM stably existed in the primary tumor site over 72 h, which consequently resulted in efficiently inhibiting the lung metastasis in breast and melanoma tumor models. The inhibition rates in two tumor models were 82.3% and 50.0%, respectively. This in vivo self-assembly strategy could be widely utilized to design effective drug-free biomaterials for inhibiting the tumor invasion and metastasis.

An in Situ Intracellular Self-Assembly Strategy for Quantitatively and Temporally Monitoring Autophagy.

Autophagy plays a crucial role in the metabolic process. So far, conventional methods are incapable of rapid, precise, and real-time monitoring of autophagy in living objects. Herein, we describe an in situ intracellular self-assembly strategy for quantitative and temporal determination of autophagy in living objectives. The intelligent building blocks (DPBP) are composed by a bulky dendrimer as a carrier, a bis(pyrene) derivative (BP) as a signal molecule, and a peptide linker as a responsive unit that can be cleaved by an autophagy-specific enzyme, i.e., ATG4B. DPBP maintains the quenched fluorescence with monomeric BP. However, the responsive peptide is specifically tailored upon activation of autophagy, resulting in self-aggregation of BP residues which emit a 30-fold enhanced fluorescence. By measuring the intensity of fluorescent signal, we are able to quantitatively evaluate the autophagic level. In comparison with traditional techniques, such as TEM, Western blot, and GFP-LC3, the reliability and accuracy of this method are finally validated. We believe this in situ intracellular self-assembly strategy provides a rapid, effective, real-time, and quantitative method for monitoring autophagy in living objects, and it will be a useful tool for autophagy-related fundamental and clinical research.

Polymeric nanoparticles promote macrophage reversal from M2 to M1 phenotypes in the tumor microenvironment.

Immunotherapy has shown promising treatment effects for a variety of cancers. However, the immune treatment efficiency for solid tumors is limited owing to insufficient infiltration of immune cells into solid tumors. The conversion of tumor-supportive macrophages to tumor-suppressive macrophages, inducing the functional reversal of macrophages, is an effective method and contributes to a subsequent antitumor response. The current challenge in the field is the poor distribution and systemic side effects associated with the use of cytokines. As a solution to this issue, we designed and synthesized microenvironment-responsive nanoparticles (P) with IL-12 payload (IL-12⊂P1). These nanoparticles could promote the systemic administration and release of IL-12 in the tumor microenvironment, and the locally responsive property of IL-12⊂P1 could subsequently re-educate tumor-associated macrophages (TAMs). In particular, our results illustrated the great therapeutic effects derived from the functional conversion of macrophages. Our strategy was to design a microenvironment-responsive material for local macrophage modification to overcome the physiological barrier of solid tumors. The shifting of macrophage phenotypes via IL-12⊂P1 achieved immunomodulation in the microenvironment for cancer therapy, with negligible cytotoxicity. We expect that the functional regulation of TAMs by pH-responsive nanomaterials is a promising therapeutic approach for cancer immunotherapy.

NIR Light Propulsive Janus-like Nanohybrids for Enhanced Photothermal Tumor Therapy.

Au-BP7@SP nanohybrids with active motion under NIR laser irradiation can effectively enhance the temperature of tumor potentially by converting the kinetic energy to thermal energy, enhancing the killing efficiency of the tumor cells compared with Au@SP. The study provides an insight of nanohybrids' effect on photothermal treatment and opens a new avenue to cancer treatment by using self-propulsion Janus nanohybrids.