Photodynamically Tumor Vessel Destruction Amplified Tumor Targeting of Nanoparticles for Efficient Chemotherapy.

Efficient tumor-targeted drug delivery is still a challenging and currently unbreakable bottleneck in chemotherapy for tumors. Nanomedicines based on passive or active targeting strategy have not yet achieved convincing chemotherapeutic benefits in the clinic due to the tumor heterogeneity. Inspired by the efficient inflammatory-cell recruitment to acute clots, we constructed a two-component nanosystem, which is composed of an RGD-modified pyropheophorbide-a (Ppa) micelle (PPRM) that mediates the tumor vascular-targeted photodynamic reaction to activate local coagulation and subsequently transmits the coagulation signals to the circulating clot-targeted CREKA peptide-modified camptothecin (CPT)-loaded nanodiscs (CCNDs) for amplifying tumor targeting. PPRM could effectively bind with the tumor vasculature and induce sufficient local thrombus by a photodynamic reaction. Local photodynamic reaction-induced tumor target amplification greatly increased the tumor accumulation of CCND by 4.2 times, thus significantly enhancing the chemotherapeutic efficacy in the 4T1 breast tumor model. In other words, this study provides a powerful platform to amplify tumor-specific drug delivery by taking advantage of the efficient crosstalk between the PPRM-activated coagulation cascade and clot-targeted CCND.

Heritable CRISPR Mutagenesis of Essential Maternal Effect Genes as a Simple Tool for Sustained Population Suppression of Invasive Species in a Zebrafish Model.

Invasive species control is important for ecological and agricultural management. Genetic methods can provide species specificity for population control. We developed heritable maternal effect embryo lethality (HMEL), a novel strategy allowing negative population pressure from HMEL individuals to be transmitted within a population across generations. We demonstrate the HMEL technique in zebrafish through genome-integrated CRISPR/Cas targeted mutagenic disruption of nucleoplasmin 2b (npm2b), a female-specific essential maternal effect gene, causing heritable sex-limited disruption of reproduction. HMEL-induced high-efficiency mutation of npm2b in females suppresses population, while males transmit the HMEL allele across generations. HMEL could be easily modified to target other genes causing sex-specific sterility, or generalized to control invasive fish or other vertebrate species for environmental conservation or agricultural protection.

Manipulating Redox Homeostasis of Cancer Stem Cells Overcome Chemotherapeutic Resistance through Photoactivatable Biomimetic Nanodiscs.

Tumor heterogeneity remains a significant obstacle in cancer therapy due to diverse cells with varying treatment responses. Cancer stem-like cells (CSCs) contribute significantly to intratumor heterogeneity, characterized by high tumorigenicity and chemoresistance. CSCs reside in the depth of the tumor, possessing low reactive oxygen species (ROS) levels and robust antioxidant defense systems to maintain self-renewal and stemness. A nanotherapeutic strategy is developed using tumor-penetrating peptide iRGD-modified high-density lipoprotein (HDL)-mimetic nanodiscs (IPCND) that ingeniously loaded with pyropheophorbide-a (Ppa), bis (2-hydroxyethyl) disulfide (S-S), and camptothecin (CPT) by synthesizing two amphiphilic drug-conjugated sphingomyelin derivatives. Photoactivatable Ppa can generate massive ROS which as intracellular signaling molecules effectively shut down self-renewal and trigger differentiation of the CSCs, while S-S is utilized to deplete GSH and sustainably imbalance redox homeostasis by reducing ROS clearance. Simultaneously, the depletion of GSH is accompanied by the release of CPT, which leads to subsequent cell death. This dual strategy successfully disturbed the redox equilibrium of CSCs, prompting their differentiation and boosting the ability of CPT to kill CSCs upon laser irradiation. Additionally, it demonstrated a synergistic anti-cancer effect by concurrently eliminating therapeutically resistant CSCs and bulk tumor cells, effectively suppressing tumor growth in CSC-enriched heterogeneous colon tumor mouse models.

Pyroptosis Induction with Nanosonosensitizer-Augmented Sonodynamic Therapy Combined with PD-L1 Blockade Boosts Efficacy against Liver Cancer.

Induction of pyroptosis can promote anti-PD-L1 therapeutic efficacy due to the release of pro-inflammatory cytokines, but current approaches can cause off target toxicity. Herein, a phthalocyanine-conjugated mesoporous silicate nanoparticle (PMSN) is designed for amplifying sonodynamic therapy (SDT) to augment oxidative stress and induce robust pyroptosis in tumors. The sub-10 nm diameter structure and c(RGDyC)-PEGylated modification enhance tumor targeting and renal clearance. The unique porous architecture of PMSN doubles ROS yield and enhances pyroptotic cell populations in tumors (25.0%) via a cavitation effect. PMSN-mediated SDT treatment efficiently reduces tumor mass and suppressed residual tumors in treated and distant sites by synergizing with PD-L1 blockade (85.93% and 77.09%, respectively). Furthermore, loading the chemotherapeutic, doxorubicin, into PMSN intensifies SDT-pyroptotic effects and increased efficacy. This is the first report of the use of SDT regimens to induce pyroptosis in liver cancer. This noninvasive and effective strategy has potential for clinical translation.

Ultrasound contrast agents from microbubbles to biogenic gas vesicles.

Microbubbles have been the earliest and most widely used ultrasound contrast agents by virtue of their unique features: such as non-toxicity, intravenous injectability, ability to cross the pulmonary capillary bed, and significant enhancement of echo signals for the duration of the examination, resulting in essential preclinical and clinical applications. The use of microbubbles functionalized with targeting ligands to bind to specific targets in the bloodstream has further enabled ultrasound molecular imaging. Nevertheless, it is very challenging to utilize targeted microbubbles for molecular imaging of extravascular targets due to their size. A series of acoustic nanomaterials have been developed for breaking free from this constraint. Especially, biogenic gas vesicles, gas-filled protein nanostructures from microorganisms, were engineered as the first biomolecular ultrasound contrast agents, opening the door for more direct visualization of cellular and molecular function by ultrasound imaging. The ordered protein shell structure and unique gas filling mechanism of biogenic gas vesicles endow them with excellent stability and attractive acoustic responses. What's more, their genetic encodability enables them to act as acoustic reporter genes. This article reviews the upgrading progresses of ultrasound contrast agents from microbubbles to biogenic gas vesicles, and the opportunities and challenges for the commercial and clinical translation of the nascent field of biomolecular ultrasound.

Manipulating Neovasculature-Targeting Capability of Biomimetic Nanodiscs for Synergistic Photoactivatable Tumor Infarction and Chemotherapy.

Tumor infarction therapy is a promising antitumor strategy with the advantages of taking a short therapy duration, less risk of resistance, and effectiveness against a wide range of tumor types. However, its clinical application is largely hindered by tumor recurrence in the surviving rim and the potential risk of thromboembolic events due to nonspecific vasculature targeting. Herein, a neovasculature-targeting synthetic high-density lipoprotein (sHDL) nanodisc loaded with pyropheophorbide-a and camptothecin (CPN) was fabricated for photoactivatable tumor infarction and synergistic chemotherapy. By manipulating the anisotropy in ligand modification of sHDL nanodiscs, CPN modified with neovaculature-targeting peptide on the planes (PCPN) shows up to 7-fold higher cellular uptake compared with that around the edge (ECPN). PCPN can efficiently bind to endothelial cells of tumor vessels, and upon laser irradiation, massive local thrombus can be induced by the photodynamic reaction to deprive nutrition supply. Meanwhile, CPT could be released in response to the tumor reductive environment, thus killing residual tumor cells in the surviving rim to inhibit recurrence. These findings not only offer a powerful approach of synergistic cancer therapy but also suggest the potential of plane-modified sHDL nanodiscs as a versatile drug delivery nanocarrier.

Biogenic Imaging Contrast Agents.

Imaging contrast agents are widely investigated in preclinical and clinical studies, among which biogenic imaging contrast agents (BICAs) are developing rapidly and playing an increasingly important role in biomedical research ranging from subcellular level to individual level. The unique properties of BICAs, including expression by cells as reporters and specific genetic modification, facilitate various in vitro and in vivo studies, such as quantification of gene expression, observation of protein interactions, visualization of cellular proliferation, monitoring of metabolism, and detection of dysfunctions. Furthermore, in human body, BICAs are remarkably helpful for disease diagnosis when the dysregulation of these agents occurs and can be detected through imaging techniques. There are various BICAs matched with a set of imaging techniques, including fluorescent proteins for fluorescence imaging, gas vesicles for ultrasound imaging, and ferritin for magnetic resonance imaging. In addition, bimodal and multimodal imaging can be realized through combining the functions of different BICAs, which helps overcome the limitations of monomodal imaging. In this review, the focus is on the properties, mechanisms, applications, and future directions of BICAs.

Major Strategies for Spatial Control of Ultrasound-Driven Gene Expression to Enhance Therapeutic Specificity.

A major challenge of gene therapy is to achieve highly specific transgene expression in tissues of interest with minimized off-target expression. Ultrasound in combination with microbubbles can transiently increase permeability of desired cells or tissues and thereby facilitate gene transfer. This kind of ultrasound-driven transgene expression has gained increasing attention due to its deep tissue penetration and high spatiotemporal resolution. However, successful genetic manipulation in vivo with ultrasound need to well optimize various aspects involved in this process. Ultrasound parameters, microbubble dose, and gene vectors need to be optimized for highly increased transgene expression in the cells of interest. Conversely, the potential off-target transgene expression and toxicities need to be reduced by modification of gene vectors and/or promoter sequence. This review will discuss some major strategies for enhanced specificity of the ultrasound-mediated gene transfer in vivo. Five major strategies will be discussed, including the integration of real-time imaging methods, local injection, targeted microbubbles loaded with nucleic acids, stealth nanocarriers, and cell-specific promoter. The advantages and limitations of each strategy were outlined, hoping to provide a guideline for researchers in achieving high specific ultrasound-driven gene expression.

Enhancing Photodynamic Therapy Efficacy Against Cancer Metastasis by Ultrasound-Mediated Oxygen Microbubble Destruction to Boost Tumor-Targeted Delivery of Oxygen and Renal-Clearable Photosensitizer Micelles.

Hypoxic tumor microenvironment and nonspecific accumulation of photosensitizers are two key factors that limit the efficacy of photodynamic therapy (PDT). Herein, a strategy of oxygen microbubbles (MBs) boosting photosensitizer micelles is developed to enhance PDT efficacy and inhibit tumor metastasis by self-assembling renal-clearable ultrasmall poly(ethylene glycol)-modified protoporphyrin IX micelles (PPM) and perfluoropentane (PFP)-doped oxygen microbubbles (OPMBs), followed by ultrasound imaging-guided OPMB destruction to realize the tumor-targeted delivery of PPM and oxygen in tumor. Doping PFP into oxygen MBs increases the production of MBs and stability of oxygen MBs, allowing for persistent circulation in blood. Following co-injection, destruction of OPMBs with ultrasound leads to ∼2.2-fold increase of tumor-specific PPM accumulation. Furthermore, the burst release of oxygen by MB destruction improves tumor oxygenation from 22 to 50%, which not only raises the production of singlet oxygen but also significantly reduces the expression of hypoxia-inducible factor-1 alpha and related genes, thus preventing angiogenesis and epithelial-mesenchymal transition. It is verified that this strategy effectively eradicates orthotopic breast cancer and inhibits lung metastasis. Furthermore, the survival rate of mice bearing orthotopic pancreatic tumor is significantly extended by such interventional PDT strategy. Therefore, the combination of ultrasmall PPM and OPMBs represents a simple but effective strategy in overcoming the limitations of PDT.

Asymmetric, amphiphilic RGD conjugated phthalocyanine for targeted photodynamic therapy of triple negative breast cancer.

Targeted photodynamic therapy (TPDT) is considered superior to conventional photodynamic therapy due to the enhanced uptake of photosensitizers by tumor cells. In this paper, an amphiphilic and asymmetric cyclo-Arg-Gly-Asp-d-Tyr-Lys(cRGDyK)-conjugated silicon phthalocyanine (RSP) was synthesized by covalently attaching the tripeptide Arg-Gly-Asp (RGD) to silicone phthalocyanine in the axial direction for TPDT of triple-negative breast cancer (TNBC). RSP was characterized by spectroscopy as a monomer in physiological buffer. Meanwhile, the modification of RSP with RGD led to a high accumulation of the photosensitizer in TNBC cells overexpressing ανß3 integrin receptors which can bind RGD, greatly reducing the risk of phototoxicity. In vitro photodynamic experiments showed that the IC50 of RSP was 295.96 nM in the 4T1 cell line, which caused significant apoptosis of the tumor cells. The tumor inhibition rate of RSP on the orthotopic murine TNBC achieved 74%, while the untargeted photosensitizer exhibited no obvious tumor inhibition. Overall, such novel targeted silicon phthalocyanine has good potential for clinical translation due to its simple synthesis route, strong targeting, and high therapeutic efficacy for TPDT treatment of TNBC.

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell (EC) gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified kallikrein-related peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial ECs, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured ECs. Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs, as determined by NFκB activation, expression of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, and monocyte adhesion. Furthermore, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

Ultrasmall porphyrin-silica core-shell dots for enhanced fluorescence imaging-guided cancer photodynamic therapy.

Clinically used small-molecular photosensitizers (PSs) for photodynamic therapy (PDT) share similar disadvantages, such as the lack of selectivity towards cancer cells, short blood circulation time, life-threatening phototoxicity, and low physiological solubility. To overcome such limitations, the present study capitalizes on the synthesis of ultra-small hydrophilic porphyrin-based silica nanoparticles (core-shell porphyrin-silica dots; PSDs) to enhance the treatment outcomes of cancer via PDT. These ultra-small PSDs, with a hydrodynamic diameter less than 7 nm, have an excellent aqueous solubility in water (porphyrin; TPPS3-NH2) and enhanced tumor accumulation therefore exhibiting enhanced fluorescence imaging-guided PDT in breast cancer cells. Besides ultra-small size, such PSDs also displayed an excellent biocompatibility and negligible dark cytotoxicity in vitro. Moreover, PSDs were also found to be stable in other physiological solutions as a function of time. The fluorescence imaging of porphyrin revealed a prolonged residence time of PSDs in tumor regions, reduced accumulation in vital organs, and rapid renal clearance upon intravenous injection. The in vivo study further revealed reduced tumor growth in 4T1 tumor-bearing bulb mice after laser irradiation explaining the excellent photodynamic therapeutic efficacy of ultra-small PSDs. Thus, ultrasmall hydrophilic PSDs combined with excellent imaging-guided therapeutic abilities and renal clearance behavior represent a promising platform for cancer imaging and therapy.

Enhancing Cancer Immunotherapeutic Efficacy with Sonotheranostic Strategies.

Immunotherapy has revolutionized the modality for establishing a firm immune response and immunological memory. However, intrinsic limitations of conventional low responsive poor T cell infiltration and immune related adverse effects urge the coupling of cancer nanomedicines with immunotherapy for boosting antitumor response under ultrasound (US) sensitization to mimic dose-limiting toxicities for safe and effective therapy against advanced cancer. US is composed of high-frequency sound waves that mediate targeted spatiotemporal control over release and internalization of the drug. The unconventional US triggered immunogenic nanoengineered arena assists the limited immunogenic dose, limiting toxicities and efficacies. In this Review, we discuss current prospects of enhanced immunotherapy using nanomedicine under US. We highlight how nanotechnology designs and incorporates nanomedicines for the reprogramming of systematic immunity in the tumor microenvironment. We also emphasize the mechanical and biological potential of US, encompassing sonosensitizer activation for enhanced immunotherapeutic efficacies. Finally, the smartly converging combinational platform of US stimulated cancer nanomedicines for amending immunotherapy is summarized. This Review will widen scientists' ability to explore and understand the limiting factors for combating cancer in a precisely customized way.

Complementing Cancer Photodynamic Therapy with Ferroptosis through Iron Oxide Loaded Porphyrin-Grafted Lipid Nanoparticles.

Nanomaterials that combine multimodality imaging and therapeutic functions within a single nanoplatform have drawn extensive attention for molecular medicines and biological applications. Herein, we report a theranostic nanoplatform based on a relatively smaller (<20 nm) iron oxide loaded porphyrin-grafted lipid nanoparticles (Fe3O4@PGL NPs). The amphiphilic PGL easily self-assembled on the hydrophobic exterior surface of ultrasmall Fe3O4 NPs, resulting in a final ultrasmall Fe3O4@PGL NPs with diameter of ∼10 nm. The excellent self-assembling nature of the as-synthesized PGL NPs facilitated a higher loading of porphyrins, showed a negligible dark toxicity, and demonstrated an excellent photodynamic effect against HT-29 cancer cells in vitro. The in vivo experimental results further confirmed that Fe3O4@PGL NPs were ideally qualified for both the fluorescence and magnetic resonance (MR) imaging guided nanoplatforms to track the biodistribution and therapeutic responses of NPs as well as to simultaneously trigger the generation of highly cytotoxic reactive oxygen species (ROS) necessary for excellent photodynamic therapy (PDT). After recording convincing therapeutic responses, we further evaluated the ability of Fe3O4@PGL NPs/Fe3O4@Lipid NPs for ferroptosis therapy (FT) via tumor microenvironment (TME) modulation for improved anticancer activity. We hypothesized that tumor-associated macrophages (TAMs) could significantly improve the efficacy of FT by accelerating the Fenton reaction in vitro. In our results, the Fe ions released in vitro directly contributed to the Fenton reaction, whereas the presence of RAW 264.7 macrophages further accelerated the ROS generation as observed by the fluorescence imaging. The significant increase in the ROS during the coincubation of NPs, endocytosed by HT-29 cells and RAW264.7 cells, further induced increased cellular toxicity of cancer cells.

Optimization of microbubble-based DNA vaccination with low-frequency ultrasound for enhanced cancer immunotherapy.

Immunotherapy is an important cancer treatment strategy; nevertheless, the lack of robust immune cell infiltration in the tumor microenvironment remains a factor in limiting patient response rates. In vivo gene delivery protocols can amplify immune responses and sensitize tumors to immunotherapies, yet non-viral transfection methods often sacrifice transduction efficiency for improved safety tolerance. To improve transduction efficiency, we optimized a strategy employing low ultrasound transmission frequency-induced bubble oscillation to introduce plasmids into tumor cells. Differential centrifugation isolated size-specific microbubbles. The diameter of the small microbubble population was 1.27 ± 0.89 µm and that of larger population was 4.23 ± 2.27 µm. Upon in vitro insonation with the larger microbubble population, 29.7% of cancer cells were transfected with DNA plasmids, higher than that with smaller microbubbles (18.9%, P <0.05) or positive control treatments with a commercial transfection reagent (12%, P < 0.01). After 48 h, gene expression increased more than two-fold in tumors treated with large, as compared with small, microbubbles. Furthermore, the immune response, including tumor infiltration of CD8+ T cells and F4/80+ macrophages, was enhanced. We believe that this safe and efficacious method can improve preclinical procedures and outcomes for DNA vaccines in cancer immunotherapy in the future.

Synergies between therapeutic ultrasound, gene therapy and immunotherapy in cancer treatment.

Due to the ease of use and excellent safety profile, ultrasound is a promising technique for both diagnosis and site-specific therapy. Ultrasound-based techniques have been developed to enhance the pharmacokinetics and efficacy of therapeutic agents in cancer treatment. In particular, transfection with exogenous nucleic acids has the potential to stimulate an immune response in the tumor microenvironment. Ultrasound-mediated gene transfection is a growing field, and recent work has incorporated this technique into cancer immunotherapy. Compared with other gene transfection methods, ultrasound-mediated gene transfection has a unique opportunity to augment the intracellular uptake of nucleic acids while safely and stably modulating the expression of immunostimulatory cytokines. The development and commercialization of therapeutic ultrasound systems further enhance the potential translation. In this Review, we introduce the underlying mechanisms and ongoing preclinical studies of ultrasound-based techniques in gene transfection for cancer immunotherapy. Furthermore, we expand on aspects of therapeutic ultrasound that impact gene therapy and immunotherapy, including tumor debulking, enhancing cytokines and chemokines and altering nanoparticle pharmacokinetics as these effects of ultrasound cannot be fully dissected from targeted gene therapy. We finally explore the outlook for this rapidly developing field.