Cryo-shocked tumor cells deliver CRISPR-Cas9 for lung cancer regression by synthetic lethality.

Although CRISPR-mediated genome editing holds promise for cancer therapy, inadequate tumor targeting and potential off-target side effects hamper its outcomes. In this study, we present a strategy using cryo-shocked lung tumor cells as a CRISPR-Cas9 delivery system for cyclin-dependent kinase 4 (CDK4) gene editing, which initiates synthetic lethal in KRAS-mutant non-small cell lung cancer (NSCLC). By rapidly liquid nitrogen shocking, we effectively eliminate the pathogenicity of tumor cells while preserving their structure and surface receptor activity. This delivery system enables the loaded CRISPR-Cas9 to efficiently target to lung through the capture in pulmonary capillaries and interactions with endothelial cells. In a NSCLC-bearing mouse model, the drug accumulation is increased nearly fourfold in lung, and intratumoral CDK4 expression is substantially down-regulated compared to CRISPR-Cas9 lipofectamine nanoparticles administration. Furthermore, CRISPR-Cas9 editing-mediated CDK4 ablation triggers synthetic lethal in KRAS-mutant NSCLC and prolongs the survival of mice.

Rationally designed approaches to augment CAR-T therapy for solid tumor treatment.

Chimeric antigen receptor T cell denoted as CAR-T therapy has realized incredible therapeutic advancements for B cell malignancy treatment. However, its therapeutic validity has yet to be successfully achieved in solid tumors. Different from hematological cancers, solid tumors are characterized by dysregulated blood vessels, dense extracellular matrix, and filled with immunosuppressive signals, which together result in CAR-T cells' insufficient infiltration and rapid dysfunction. The insufficient recognition of tumor cells and tumor heterogeneity eventually causes cancer reoccurrences. In addition, CAR-T therapy also raises safety concerns, including potential cytokine release storm, on-target/off-tumor toxicities, and neuro-system side effects. Here we comprehensively review various targeting aspects, including CAR-T cell design, tumor modulation, and delivery strategy. We believe it is essential to rationally design a combinatory CAR-T therapy via constructing optimized CAR-T cells, directly manipulating tumor tissue microenvironments, and selecting the most suitable delivery strategy to achieve the optimal outcome in both safety and efficacy.

Local Drug Delivery Techniques for Triggering Immunogenic Cell Death.

Immunogenic cell death (ICD), a dying state of the cells, encompasses the changes in the conformations of cell surface and the release of damage-associated molecular patterns, which could initiate an adaptive immune response by stimulating the dendritic cells to present antigens to T cells. Advancements in biomaterials, nanomedicine, and micro- and nano-technologies have facilitated the development of effective ICD inducers, but the potential toxicity of these vesicles encountered in drug delivery via intravenous administration hampers their further application. As alternatives, the local drug delivery systems have gained emerging attention due to their ability to prolong the retention of high payloads at the lesions, sequester drugs from harsh environments, overcome biological barriers to exert optimal efficacy, and minimize potential side effects to guarantee bio-safety. Herein, a brief overview of the local drug delivery techniques used for ICD inducers is provided, explaining how these techniques broaden, alter, and enhance the therapeutic capability while circumventing systemic toxicity at the same time. The historical context and prominent examples of the local administration of ICD inducers are introduced. The complexities, potential pitfalls, and opportunities for local drug delivery techniques in cancer immunotherapy are also discussed.

Adipocytes Encapsulating Telratolimod Recruit and Polarize Tumor-Associated Macrophages for Cancer Immunotherapy.

Tumor-associated adipocytes (TAAs) recruit monocytes and promote their differentiation into tumor-associated macrophages (TAMs) that support tumor development. Here, TAAs are engineered to promote the polarization of TAMs to the tumor suppressive M1 phenotype. Telratolimod, a toll-like receptor 7/8 agonist, is loaded into the lipid droplets of adipocytes to be released at the tumor site upon tumor cell-triggered lipolysis. Locally administered drug-loaded adipocytes increased tumor suppressive M1 macrophages in both primary and distant tumors and suppressed tumor growth in a melanoma model. Furthermore, drug-loaded adipocytes improved CD8+ T cell-mediated immune responses within the tumor microenvironment and favored dendritic cell maturation in the tumor draining lymph nodes.

CRISPR System-Linked Self-Assembling Nanoplatforms for Inspection and Screening of Gastric Cancer Stem Cells.

Cancer stem cells (CSCs) possess a high degree of plasticity, constituting a formidable challenge to identify and screen CSCs in situ with outstanding specificity and sensitivity. To overcome this limitation, a self-assembled heterodimer consisting of clustered regularly interspaced short palindromic repeats/Cas12a (named A-CCA) linkage is designed for in situ identification and screening of gastric CSCs (GCSCs) from gastric cancer cells (GCCs). In this system, the editable character of crRNA performs recognition of dual-targets in GCSCs, effectively boosting the specificity of identification, while the enzymatic reaction of Cas12a contributes meaningfully to the sensitivity of sensing, enabling in situ examination and screening of GCSCs. Specifically, the A-CCA nanoplatforms hybridized with ABCG 2 and ABCB 1 overexpress in GCSCs, which can generate heterodimers and simultaneously restore the function of trans-cleavage. At this time, the asymmetry of the heterodimer causes a circular dichroism signal, which together with the recovered fluorescence signal form a dual-signals output system that can further ensure the precision of screening GCSC. Therefore, fluorescence-enhanced GCSCs can be sorted out from GCCs by flow cytometry. Furthermore, GCSCs screened by this assay possess extremely aggressive tumorigenic efficiency, providing a fundamental research object for further developing CSC targeted drugs in vivo.

Salicylic acid-based nanomedicine with self-immunomodulatory activity facilitates microRNA therapy for metabolic skeletal disorders.

Metabolic skeletal disorders remain a major clinical challenge. The complexity of this disease requires a strategy to address the net effects of both inflammation and impaired bone formation. microRNA-based gene therapy provides several therapeutic advantages to tackle these issues. Herein, we describe a microRNA-21 (miR-21) delivery system with an additional therapeutic effect from that of the delivery carrier itself. Poly (salicylic acid) (PSA) is, for the first time, synthesized via polycondensation of salicylic acid (SA), a bioactive ingredient widely used for anti-inflammation in medicine. PSA can self-assemble into nanoparticles (PSA-NPs) and can effectively deliver genes both in vitro and in vivo. The carrier was then attached to repetitive sequences of aspartate, serine, serine (DSS)6 for delivering miRNAs specifically to bone-formation surfaces. In vitro studies showed that miR-21@PSA-NP could effectively realize the intracellular delivery of miR-21 with low toxicity, while in vivo results indicated that the miR-21@PSA-NP-DSS6 prolonged blood circulation time, enhanced bone accumulation, and significantly improved the efficacy of miR-21-based bone anabolic therapy in osteoporotic mice. The constructed delivery system (miR-21@PSA-NP-DSS6) inherited the advantages of both SA and miR-21, which could ameliorate bone-inflamed niche and rescued the impaired bone formation ability. The synergy of anti-inflammatory and pro-osteogenic effects significantly improved trabecular bone microstructure in osteoporotic mice. STATEMENT OF SIGNIFICANCE: The complexity of metabolic skeletal disorders requires a strategy to address the net effects of both inflammation and impaired bone formation. microRNA-based gene therapy provides several therapeutic advantages to tackle these issues. We develop a novel microRNA-21 delivery system with additional therapeutic effect from that of the gene carrier itself. Poly (salicylic acid) (PSA) nanoparticles, for the first time, synthesized via polycondensation of salicylic acid and can effectively deliver genes both in vitro and in vivo. The constructed delivery system (miR-21@PSA-NP-DSS6) inherited the advantages of both SA (commonly used anti-inflammation drug in medicine) and miR-21 (a pro-osteogenic molecule), which could ameliorate bone-inflamed niche, rescued impaired bone formation ability and significantly improved trabecular bone microstructure in osteoporotic mice.

Adipocyte-Derived Anticancer Lipid Droplets.

Engineering of efficient and safe materials remains a challenge for cancer therapy. Here, the lipid droplet, an organelle in adipocytes, is demonstrated to be a controllable and biocompatible vehicle to deliver anticancer drugs. It is validated that isolated lipid droplets maintain their key physiological functions to interact with other organelles and augment the therapeutic effect of cancer photodynamic therapy by encapsulation with a lipid-conjugated photosensitizer (Pyrolipid) through a variety of pathways, including generation of reactive oxygen species (ROS); lipid peroxidation; and endoplasmic reticulum (ER) stress. As such, the IC50 value of Pyrolipid is reduced by 6.0-fold when loaded into the lipid droplet. Of note, in vivo results demonstrate that engineered lipid droplets induce significant inhibition of tumor growth with minimal side effects.

Adapting and Remolding: Orchestrating Tumor Microenvironment Normalization with Photodynamic Therapy by Size Transformable Nanoframeworks.

Abnormal tumor microenvironment (TME) facilitates tumor proliferation and metastasis and establishes physiological barriers for effective transport of therapeutics inside the tumor, posing great challenges for cancer treatment. We designed a core-satellite size transformable nanoframework (denoted as T-PFRT) that can synchronously adapt to and remold TME for augmenting photodynamic therapy to inhibit tumor growth and prevent tumor metastasis. Upon matrix metalloproteinase 2 (MMP2)-responsive dissociation of the nanoframework in TME, the core structure loaded with TGFß signaling pathway inhibitor and oxygen-carrying hemoglobin aims to stroma remodeling and hypoxia relief, allowing photosensitizer-encapsulated satellite particles to penetrate to deep-seated tumor for oxygen-fueled photodynamic therapy. T-PFRT could overcome the stroma and hypoxia barriers for delivering therapeutics and gain excellent therapeutic outcomes in the treatment of primary and metastatic tumors.

Roadmap on nanomedicine.

Since the launch of the Alliance for Nanotechnology in Cancer by the National Cancer Institute in late 2004, several similar initiatives have been promoted all over the globe with the intention of advancing the diagnosis, treatment and prevention of cancer in the wake of nanoscience and nanotechnology. All this has encouraged scientists with diverse backgrounds to team up with one another, learn from each other, and generate new knowledge at the interface between engineering, physics, chemistry and biomedical sciences. Importantly, this new knowledge has been wisely channeled towards the development of novel diagnostic, imaging and therapeutic nanosystems, many of which are currently at different stages of clinical development. This roadmap collects eight brief articles elaborating on the interaction of nanomedicines with human biology; the biomedical and clinical applications of nanomedicines; and the importance of patient stratification in the development of future nanomedicines. The first article reports on the role of geometry and mechanical properties in nanomedicine rational design; the second articulates on the interaction of nanomedicines with cells of the immune system; and the third deals with exploiting endogenous molecules, such as albumin, to carry therapeutic agents. The second group of articles highlights the successful application of nanomedicines in the treatment of cancer with the optimal delivery of nucleic acids, diabetes with the sustained and controlled release of insulin, stroke by using thrombolytic particles, and atherosclerosis with the development of targeted nanoparticles. Finally, the last contribution comments on how nanomedicine and theranostics could play a pivotal role in the development of personalized medicines. As this roadmap cannot cover the massive extent of development of nanomedicine over the past 15 years, only a few major achievements are highlighted as the field progressively matures from the initial hype to the consolidation phase.

A programmable polymer library that enables the construction of stimuli-responsive nanocarriers containing logic gates.

Stimuli-responsive biomaterials that contain logic gates hold great potential for detecting and responding to pathological markers as part of clinical therapies. However, a major barrier is the lack of a generalized system that can be used to easily assemble different ligand-responsive units to form programmable nanodevices for advanced biocomputation. Here we develop a programmable polymer library by including responsive units in building blocks with similar structure and reactivity. Using these polymers, we have developed a series of smart nanocarriers with hierarchical structures containing logic gates linked to self-immolative motifs. Designed with disease biomarkers as inputs, our logic devices showed site-specific release of multiple therapeutics (including kinase inhibitors, drugs and short interfering RNA) in vitro and in vivo. We expect that this 'plug and play' platform will be expanded towards smart biomaterial engineering for therapeutic delivery, precision medicine, tissue engineering and stem cell therapy.

Promoting Oxidative Stress in Cancer Starvation Therapy by Site-Specific Startup of Hyaluronic Acid-Enveloped Dual-Catalytic Nanoreactors.

Cutting off the glucose supply by glucose oxidase (GOx) has been regarded as an emerging strategy in cancer starvation therapy. However, the standalone GOx delivery suffered suboptimal potency for tumor elimination and potential risks of damaging vasculatures and normal organs during transportation. To enhance therapeutic efficacy and tumor specificity, a site-specific activated dual-catalytic nanoreactor was herein constructed by embedding GOx and ferrocene in hyaluronic acid (HA)-enveloped dendritic mesoporous silica nanoparticles to promote intratumoral oxidative stress in cancer starvation. In this nanoreactor, the encapsulated GOx served as the primary catalyst that accelerated oxidation of glucose and generation of H2O2, while the covalently linked ferrocene worked as the secondary catalyst for converting the upstream H2O2 to more toxic hydroxyl radicals (•OH) via a classic Fenton reaction. The outmost HA shell not only offered a shielding layer for preventing blood glucose from oxidation during nanoreactor transportation, thus minimizing the probable oxidative damage to normal tissues, but also imparted the nanoreactor with targeting ability for facilitating its internalization into CD44-overexpressing tumor cells. After the nanoreactor was endocytosed by target cells, the HA shell underwent hyaluronidase-triggered degradation in lysosomes and switched on the cascade catalytic reaction mediated by GOx and ferrocene. The resulting glucose exhaustion and •OH accumulation would effectively kill cancer cells and suppress tumor growth via combination of starvation and oxidative stress enhancement. Both in vitro and in vivo results indicated the significantly amplified therapeutic effects of this synergistic therapeutic strategy based on the dual-catalytic nanoreactor. Our study provides a new avenue for engineering therapeutic nanoreactors that take effect in a tumor-specific and orchestrated fashion for cancer starvation therapy.

Cascaded Aptamers-Governed Multistage Drug-Delivery System Based on Biodegradable Envelope-Type Nanovehicle for Targeted Therapy of HER2-Overexpressing Breast Cancer.

Tumor-specific therapeutic platforms with improved targeting efficacy and minimized side effect are crucial in cancer therapy. Capitalizing on the recognition capability and biocompatibility of aptamers, we herein designed a multistage targeted drug-delivery system using multiple biodegradable molecules-enveloped nanovehicle that can be employed to efficiently treat human epithelial growth factor receptor (HER2)-overexpressing breast cancer. In this nanovehicle, two aptamers respectively specific to HER2 and ATP were organized in a hierarchical manner. The outmost HER2 aptamer (HB5) governs the recognition to HER2 protein overexpressed in SK-BR-3 cell lines, while the ATP aptamer incorporated with anticancer drug (-)-epigallocatechin gallate (EGCG) and protamine sulfate in the inner core functions as a switch of drug release in response to abundant intracellular ATP. The targeting and drug locker aptamers were cascaded for active targeting effect and stimuli responsiveness, guaranteeing the site-specific drug transportation and endogenous species-triggered drug release inside the tumor cells. Moreover, nanostructured lipid carriers (NLCs) were constructed to wrap and stabilize the loosely bounded ternary complex, minimizing premature drug leakage potentially encountered by the biomolecule assembled nanocarriers. This multiple biomolecules-enveloped nanovehicle demonstrated improved inhibitory actions on tumor growth and minimum side effect to normal organs and tissues both in vitro and in vivo. The presented nanovehicle built from recognition and therapeutic components in a nontoxic framework offered a promising drug-delivery platform with transport precision and biological safety.

"Stealth and Fully-Laden" Drug Carriers: Self-Assembled Nanogels Encapsulated with Epigallocatechin Gallate and siRNA for Drug-Resistant Breast Cancer Therapy.

For codelivery of therapeutic genes and chemical agents in combined therapy, the ideal drug delivery system entails high-capacity and low-body toxicity carriers, allowing adequate drug dose for tumor regions while yielding low residues in normal tissues. To augment the gene/drug load capacity and circumvent the potential toxicity brought by traditional inorganic and polymeric nanocarriers, a "stealth" carrier was herein designed in a simple self-assembly of (-)-epigallocatechin-3- O-gallate (EGCG) and small interfering RNA (siRNA) by recruiting protamine as a biodegradable medium for the treatment of drug-resistant triple-negative breast cancer. In the self-assembled nanogel, entrapped siRNA played a central role in sensitizing the tumor response to EGCG-involved chemotherapy, and the positively charged protamine served as the assembly skeleton to fully accommodate gene and drug molecules and minimize the factors causing side effects. As compared to stand-alone chemotherapy with EGCG, the multicomponent nanogel revealed a 15-fold increase in the cytotoxicity to drug-resistant MDA-MB-231 cell line. Moreover, equipped with hyaluronic acid and tumor-homing cell-penetrating peptide as the outmost targeting ligands, the siRNA- and EGCG-loaded nanogel demonstrates superior selectivity and tumor growth inhibition to free EGCG in xenograft MDA-MB-231 tumor-bearing mice. Meanwhile, thanks to the acknowledged biosafety of protamine, little toxicity was found to normal tissues and organs in the animal model. This gene/drug self-assembly caged in a biodegradable carrier opens up an effective and secure route for drug-resistant cancer therapy and provides a versatile approach for codelivery of other genes and drugs for different medical purposes.

FITC Doped Rattle-Type Silica Colloidal Particle-Based Ratiometric Fluorescent Sensor for Biosensing and Imaging of Superoxide Anion.

Fluorescent nanosensors have been widely applied in recognition and imaging of bioactive small molecules; however, the complicated surface modification process and background interference limit their applications in practical biological samples. Here, a simple, universal method was developed for ratiometric fluorescent determination of general small molecules. Taking superoxide anion (O2(•-)) as an example, the designed sensor was composed of three main moieties: probe carrier, rattle-type silica colloidal particles (mSiO2@hmSiO2 NPs); reference fluorophore doped into the core of NPs, fluorescein isothiocyanate (FITC); fluorescent probe for superoxide anion, hydroethidine (HE). In the absence of O2(•-), the sensor just emitted green fluorescence of FITC at 518 nm. When released HE was oxidized by O2(•-), the oxidation product exhibited red fluorescence at 570 nm and the intensity was linearly associated with the concentration of O2(•-), while that of reference element remained constant. Accordingly, ratiometric determination of O2(•-) was sensitively and selectively achieved with a linear range of 0.2-20 µM, and the detection limit was calculated as low as 80 nM. Besides, the technique was also successfully applied for dual-emission imaging of O2(•-) in live cells and realized visual recognition with obvious fluorescence color change in normal conditions or under oxidative stress. As long as appropriate reference dyes and sensing probes are selected, ratiometric biosensing and imaging of bioactive small molecules would be achieved. Therefore, the design could provide a simple, accurate, universal platform for biological applications.