Cytoskeleton-modulating nanomaterials and their therapeutic potentials.

The cytoskeleton, an intricate network of protein fibers within cells, plays a pivotal role in maintaining cell shape, enabling movement, and facilitating intracellular transport. Its involvement in various pathological states, ranging from cancer proliferation and metastasis to the progression of neurodegenerative disorders, underscores its potential as a target for therapeutic intervention. The exploration of nanotechnology in this realm, particularly the use of nanomaterials for cytoskeletal modulation, represents a cutting-edge approach with the promise of novel treatments. Inorganic nanomaterials, including those derived from gold, metal oxides, carbon, and black phosphorus, alongside organic variants such as peptides and proteins, are at the forefront of this research. These materials offer diverse mechanisms of action, either by directly interacting with cytoskeletal components or by influencing cellular signaling pathways that, in turn, modulate the cytoskeleton. Recent advancements have introduced magnetic field-responsive and light-responsive nanomaterials, which allow for targeted and controlled manipulation of the cytoskeleton. Such precision is crucial in minimizing off-target effects and enhancing therapeutic efficacy. This review explores the importance of research into cytoskeleton-targeting nanomaterials for developing therapeutic interventions for a range of diseases. It also addresses the progress made in this field, the challenges encountered, and future directions for using nanomaterials to modulate the cytoskeleton. The continued exploration of nanomaterials for cytoskeleton modulation holds great promise for advancing therapeutic strategies against a broad spectrum of diseases, marking a significant step forward in the intersection of nanotechnology and medicine.

External cold atmospheric plasma-responsive on-site hydrogel for remodeling tumor immune microenvironment.

Although immunotherapy has recently emerged as a promising anti-tumor approach, it remains limited by the immunosuppressive tumor microenvironment. Cold atmospheric plasma irradiation can generate reactive oxygen species and trigger the presentation of tumor-associated antigens. Here, we exploited cold atmospheric plasma for on-site hydrogel application in the tumor environment, aiming to facilitate the sustainable uptake of tumor-associated antigens and nanoadjuvants by dendritic cells. Hyaluronic acid-tyramine conjugate was intratumorally injected as a liquid and formed an on-site hydrogel under irradiation with cold atmospheric plasma. Intratumoral delivery of hyaluronic acid-tyramine conjugate with transforming growth factor ß-blocking nanoadjuvant (TLN) followed by cold atmospheric plasma irradiation yielded a micro-network of TLN-loaded hydrogel (TLN@CHG). In vivo intratumoral injection of TLN@CHG promoted the activation of dendritic cells and more effectively increased the proportion of CD4 T cells and CD8 T cells in the tumor microenvironment, compared to the groups receiving TLN or hydrogel alone. Moreover, in CT26 tumor model mice, cold atmospheric plasma-induced TLN@CHG therapy ablated the primary tumor and provided 100% survival among mice rechallenged with CT26 cells. Taken together, our findings suggest that an on-site hydrogel-based micro-network of TLN has the potential to remodel the tumor immune microenvironment. Although we used TLN in this study, the concept could be extended to support the sustained action of other nanoadjuvants in a hydrogel micro-network.

DNA-cloaked nanoparticles for tumor microenvironment-responsive activation.

Although progress has been made in developing tumor microenvironment-responsive delivery systems, the list of cargo-releasing stimuli remains limited. In this study, we report DNA nanothread-cloaked nanoparticles for reactive oxygen species (ROS)-rich tumor microenvironment-responsive delivery systems. ROS is well known to strongly induce DNA fragmentation via oxidative stress. As a model anticancer drug, hydrophobic omacetaxine was entrapped in branched cyclam ligand-modified nanoparticles (BNP). DNA nanothreads were prepared by rolling-circle amplification and complexed to BNP, yielding DNA nanothread-cloaked BNP (DBNP). DBNP was unmasked by DNA nanothread-degrading ROS and culture supernatants of LNCaP cells. The size and zeta potential of DBNP were changed by ROS. In ROShigh LNCaP cells, but not in ROSlow fibroblast cells, the uptake of DBNP was higher than that of other nanoparticles. Molecular imaging revealed that DBNP exhibited greater distribution to tumor tissues, compared to other nanoparticles. Ex vivo mass spectrometry-based imaging showed that omacetaxine metabolites were distributed in tumor tissues of mice treated with DBNP. Intravenous administration of DBNP reduced the tumor volume by 80% compared to untreated tumors. Profiling showed that omacetaxine treatment altered the transcriptional profile. These results collectively support the feasibility of using polymerized DNA-masked nanoparticles for selective activation in the ROS-rich tumor microenvironment.

DNA-based artificial dendritic cells for in situ cytotoxic T cell stimulation and immunotherapy.

In immunotherapy, ex vivo stimulation of T cells requires significant resources and effort. Here, we report artificial dendritic cell-mimicking DNA microflowers (DM) for programming T cell stimulation in situ. To mimic dendritic cells, DNA-based artificial dendritic microflowers were constructed, surface-coated with polydopamine, and further modified with anti-CD3 and anti-CD28 antibodies to yield antibody-modified DM (DM-A). The porous structure of DM-A allowed entrapment of the T cell-stimulating cytokine, ineterleukin-2, yielding interleukin-2-loaded DM-A (DM-AI). For comparison, polystyrene microparticles coated with polydopamine and modified with anti-CD3 and anti-CD28 antibodies (PS-A) were used. Compared to PS-A, DM-AI showed significantly greater contact with T cell surfaces. DM-AI provided the highest ex vivo expansion of cytotoxic T cells. Local injection of DM-AI to tumor tissues induced the recruitment of T cells and expansion of cytotoxic T cells in tumor microenvironments. Unlike the other groups, model animals injected with DM-AI did not exhibit growth of primary tumors. Treatment of mice with DM-AI also protected against growth of a rechallenged distant tumor, and thus prevented tumor recurrence in this model. DM-AI has great potential for programmed stimulation of CD8+ T cells. This concept could be broadly extended for the programming of specific T cell stimulation profiles.

Fibroblast activation protein activated antifibrotic peptide delivery attenuates fibrosis in mouse models of liver fibrosis.

In liver fibrosis, activated hepatic stellate cells are known to overexpress fibroblast activation protein. Here we report a targeted antifibrotic peptide-delivery system in which fibroblast activation protein, which is overexpressed in fibrotic regions of the liver, liberates the antifibrotic peptide melittin by cleaving a fibroblast activation protein-specific site in the peptide. The promelittin peptide is linked to pegylated and maleimide-functionalized liposomes, resulting in promelittin-modified liposomes. The promelittin-modified liposomes were effective in reducing the viability of activated hepatic stellate cells but not that of control cells. In three types of liver fibrosis mouse models, intravenously administered promelittin-modified liposomes significantly reduces fibrotic regions. In addition, in the bile duct ligation mouse model promelittin-modified liposome-treatment increases overall survival. Although this peptide-delivery concept was tested for liver fibrosis, it can potentially be adapted to other fibrotic diseases.

Nanocomplex-Mediated In Vivo Programming to Chimeric Antigen Receptor-M1 Macrophages for Cancer Therapy.

Chimeric antigen receptor-T (CAR-T) cell immunotherapy has shown impressive clinical outcomes for hematologic malignancies. However, its broader applications are challenged due to its complex ex vivo cell-manufacturing procedures and low therapeutic efficacy against solid tumors. The limited therapeutic effects are partially due to limited CAR-T cell infiltration to solid tumors and inactivation of CAR-T cells by the immunosuppressive tumor microenvironment. Here, a facile approach is presented to in vivo program macrophages, which can intrinsically penetrate solid tumors, into CAR-M1 macrophages displaying enhanced cancer-directed phagocytosis and anti-tumor activity. In vivo injected nanocomplexes of macrophage-targeting nanocarriers and CAR-interferon-γ-encoding plasmid DNA induce CAR-M1 macrophages that are capable of CAR-mediated cancer phagocytosis, anti-tumor immunomodulation, and inhibition of solid tumor growth. Together, this study describes an off-the-shelf CAR-macrophage therapy that is effective for solid tumors and avoids the complex and costly processes of ex vivo CAR-cell manufacturing.

Photosensitizer-Trapped Gold Nanocluster for Dual Light-Responsive Phototherapy.

Photoresponsive nanomaterials have recently received great attention in the field of cancer therapy. Here, we report a photosensitizer-trapped gold nanocluster that can facilitate dual light-responsive cancer therapy. We utilized methylene blue (MB) as a model photosensitizer, gold nanocluster as a model photothermal agent, and a polymerized DNA as the backbone of the nanocluster. We synthesized MB-intercalated gold DNA nanocluster (GMDN) via reduction and clustering of gold ions on a template consisting of MB-intercalated long DNA. Upon GMDN treatment, cancer cells revealed clear cellular uptake of MB and gold clusters; following dual light irradiation (660 nm/808 nm), the cells showed reactive oxygen species generation and increased temperature. Significantly higher cancer cell death was observed in cells treated with GMDN and dual irradiation compared with non-irradiated or single light-irradiated cells. Mice systemically injected with GMDN showed enhanced tumor accumulation compared to that of free MB and exhibited increased temperature upon near infrared irradiation of the tumor site. Tumor growth was almost completely inhibited in GMDN-treated tumor-bearing mice after dual light irradiation, and the survival rate of this group was 100% over more than 60 days. These findings suggest that GMDN could potentially function as an effective phototherapeutic for the treatment of cancer disease.

Biomimetic polymeric nanoparticle-based photodynamic immunotherapy and protection against tumor rechallenge.

In this study, we sought to design a bionanomaterial that could exert anticancer effects against primary tumors and protect against rechallenged tumors via photodynamic immunotherapy. As a biomaterial, we used an amphiphilic phenylalanine derivative of poly-gamma glutamic acid, which forms nanoparticles by self-assembly. For anticancer effects, we co-entrapped hydrophobic chlorin e6 and monophosphoryl lipid A in the core of the plain amphiphilic phenylalanine nanoparticles (AN), to generate M/C/AN. For comparison, we used plain AN and chlorin e6-loaded AN (C/AN). In vitro studies showed that B16F10 cancer cells treated with C/AN or M/C/AN generated reactive oxygen species and exhibited an enhanced surface display of calreticulin upon exposure to 660 nm light irradiation. C/AN and M/C/AN exerted similar photodynamic anticancer effects; however, M/C/AN, but not C/AN, induced in vitro dendritic cell maturation. Our biodistribution study revealed that C/AN and M/C/AN showed higher accumulation at the tumor tissues compared to that seen in the free chlorin e6-treated group. In B16F10 tumor-bearing mice, the intravenous injection of C/AN or M/C/AN showed similar photodynamic anticancer effects against primary tumors. However, the growth of rechallenged tumors was more significantly inhibited in the M/C/AN group compared to the C/AN group. At day 40 after inoculation of the primary tumor, M/C/AN-treated mice showed 100% survival, whereas the other groups showed 0% survival. In the tumor microenvironment, higher infiltration of CD8+ T cells was observed in the M/C/AN group compared to the other groups. Our results suggest that AN co-loaded with a photosensitizer and an immune stimulant may hold great potential for use in photodynamic immunotherapy to inhibit both primary and metastatic tumors.

Staphylococcus aureus-mimetic control of antibody orientation on nanoparticles.

We designed a bacterio-mimetic nanoparticle that can noncovalently control the orientation of attached antibodies. Liposomes with Fc-binding peptide (FcBP), formulated using FcBP-conjugated PEGylated lipid, were used as model nanoparticles. Compared with control nanoparticles surface-modified with antibody covalently attached via maleimide functional groups (Mal-NPs), FcBP-capped nanoparticles (FcBP-NPs) exhibited greater binding affinity to the target protein. Human epidermal growth factor receptor 2 (HER2)-specific antibody-modified FcBP-NPs (HER2/FcBP-NPs) showed 5.3-fold higher binding affinity to HER2 than isotype IgG antibody-modified NPs, and 2.6-fold higher affinity compared with anti-HER2 antibody-conjugated Mal-NPs. Cellular uptake of HER2/FcBP-NPs in HER2-positive cells was significantly higher than that of other formulations. The biodistribution of HER2/FcBP-NPs was higher than that of antibody-conjugated NPs in HER2-positive tumor tissues, but not in HER2-negative tumors. Our findings suggest the potential of bacteriomimetic nanoparticles for controlling the orientation of antibody attachment. These nanoparticles may have diverse applications in nanomedicine, including drug delivery, molecular imaging, and diagnosis.