Biomaterials

Development of an innovative extracellular vesicle mimetic delivery platform for efficient miRNA delivery to tumours

Extracellular vesicles (EVs) display high degree of tissue tropism and therefore represent promising carriers for tissue-specific delivery of genes or drugs for the treatment of human diseases. However, current approaches for the loading of therapeutics into EVs have low entrapment efficiency and also do not adequately deplete endogenous EV content; thus, more effective approaches are needed. Here, we report an innovative EXtraCElluar vesicle surface Ligand-NanoParticles (EXCEL NPs), generated by transferring moieties of EVs onto the surface of synthetic nanoparticles. EXCEL NPs facilitate the efficient entrapment of therapeutics (89 % efficiency) and are completely devoid of pre-existing unwanted EV internal content. Importantly, we show that EXCEL NPs formulated using EVs derived from endothelial cells, astrocytes and macrophages retain the delivery characteristics of the original EVs. Using miRNA-146a as a model anti-cancer therapeutic, we further demonstrated successful delivery of miRNA-146a to IG10 orthotopic ovarian tumours in immune competent mice using EXCEL NPs formulated with macrophage-derived EVs. Our findings establish a new clinically translatable approach to leverage characteristics of endogenous EVs for therapeutic delivery. The versatility of the platform enables future application to different target cell types and therapeutic modalities.

A biomimetic model of 3D fluid extracellular macromolecular crowding microenvironment fine-tunes ovarian cancer cells dissemination phenotype

An early fundamental step in ovarian cancer progression is the dissemination of cancer cells through liquid environments, one of them being cancer ascites accumulated in the peritoneal cavity. These biological fluids are highly crowded with a high total macromolecule concentration. This biophysical property of fluids is widely used in tissue engineering for a few decades now, yet is largely underrated in cancer biomimetic models. To unravel the role of fluids extracellular macromolecular crowding (MMC), we exposed ovarian cancer cells (OCC) to high molecular weight inert polymer solutions. High macromolecular composition of extracellular liquid presented a differential effect: i) it impeded non-adherent OCC aggregation in suspension and, decreased their adhesion; ii) it promoted adherent OCC migration by decreasing extracellular matrix deposition. Besides, there seemed to be a direct link between the extracellular MMC and intracellular processes, especially the actin cytoskeleton organization and the nucleus morphology. In conclusion, extracellular fluid MMC orients OCC dissemination phenotype. Integrating MMC seems crucial to produce more relevant mimetic 3D in vitro fluid models to study ovarian dissemination but also to screen drugs.

Multifunctional tumor-targeted PLGA nanoparticles delivering Pt(IV)/siBIRC5 for US/MRI imaging and overcoming ovarian cancer resistance

Cisplatin (Pt(II)) resistance is an important factor in the high mortality rates of ovarian cancer. Herein, we synthesized multifunctional tumor-targeted poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs-cRGD) for monitoring therapeutic effects by dual-mode imaging and overcoming cisplatin resistance. Uniformly sized NPs-cRGD demonstrated controlled and sustained release of drugs and genes, excellent gene loading and gene protection capacity, good storage stability and no serum-induced aggregation in vitro. NPs-cRGD demonstrated clear, targeting and prolonged ultrasound imaging and magnetic resonance imaging (MRI) in vivo. The targeting of NPs-cRGD combined with ultrasound facilitated nanoparticle penetrattion into cells; entry was time-dependent. NPs-cRGD escaped from lysosomes, thereby preventing siBIRC5 degradation, which enabled siBIRC5 to efficiently inhibit the antiapoptosis effects of BIRC5 in SKO3-DDP to overcome the antiapoptosis properties of resistant cells. Furthermore, Pt(IV) in NPs-cRGD exhausted glutathione (GSH), thereby increasing drug accumulation to effectively increase Pt(II) levels. The subsequent combination of Pt(II) with DNA prevented the expressions of genes and upregulated the expression of p53 to induce the mitochondria apoptosis pathway. The reduced GSH activity and the generation of Pt(II) further promoted high levels of reactive oxygen species (ROS) to induce cell apoptosis. Therefore, NPs-cRGD with ultrasound promoted the apoptosis of resistant ovarian cancer cells by multiple mechanisms, including increased cellular drug accumulation, reversed antiapoptotic effects by siBIRC5, and enhanced ROS levels. In a tumor-bearing nude mice model, NPs-cRGD with US demonstrated excellent tumor-targeting, high efficiency tumor inhibition and low systemic toxicity. Therefore, NPs-cRGD provides a means to monitor treatment processes and can be combined with ultrasound treatment to overcome ovarian cancer resistance in vitro and in vivo.

Calcium phosphate nanoneedle based gene delivery system for cancer genetic immunotherapy

Ovarian cancer has become one of the most common gynecological cancers with a high mortality. However, conventional surgery together with combination chemotherapy is difficult to achieve ideal therapeutic effect. Although genetic immunotherapy is applied to active immune responses against cancer, the absence of efficient in vivo gene delivery technique is still an obstacle in clinical application. To overcome these problems, a minicircle DNA vector encoding humanized anti-EpCAM/CD3 bispecific antibody (BsAb

Development of a near infrared protein nanoprobe targeting Thomsen-Friedenreich antigen for intraoperative detection of submillimeter nodules in an ovarian peritoneal carcinomatosis mouse model

The epithelial ovarian cancer is one of the most lethal gynecological malignancy due to its late diagnostic and many relapses observed after first line of treatment. Once diagnose, the most important prognostic factor is the completeness of cytoreductive surgery. To achieve this goal, surgeons have to pinpoint and remove nodules, especially the smallest nodules. Recent advances in fluorescence-guided surgery led us to develop a recombinant lectin as a nanoprobe for the microscopic detection of nodules in the peritoneal cavity of tumor-bearing mice. This lectin has an intrinsic specificity for a carcinoma-associated glycan biomarker, the Thomsen-Friedenreich antigen. In this study, after its labelling by a near infrared dye, we first demonstrated that this nanoprobe allowed indirect detection of nodules already implanted in the peritoneal cavity, through tumor microenvironment targeting. Secondly, in a protocol mimicking the scattering of cells during surgery, we obtained a direct and long-lasting detection of tumor cells in vivo. This lectin as already been described as a nanocontainer able to do targeted delivery of a therapeutic compound to carcinoma cells. Future developments will focus on the combination of the nanoprobe and nanocontainer aspects in an intraperitoneal nanotheranostic approach.

Hypoxia-responsive micelles deprive cofactor of stearoyl-CoA desaturase-1 and sensitize ferroptotic ovarian cancer therapy

Ferroptosis has been recognized as a promising therapeutic strategy for cancer due to its unique mechanism of action. However, the upregulation of stearoyl-CoA desaturase 1 (SCD1) in ovarian cancer leads to resistance to ferroptotic therapy. Zinc ion (Zn

Multifaceted cancer alleviation by cowpea mosaic virus in a bioprinted ovarian cancer peritoneal spheroid model

Ovarian cancer (OvCa) is a leading cause of mortality among gynecological malignancies and usually manifests as intraperitoneal spheroids that generate metastases, ascites, and an immunosuppressive tumor microenvironment. In this study, we explore the immunomodulatory properties of cowpea mosaic virus (CPMV) as an adjuvant immunotherapeutic agent using an in vitro model of OvCa peritoneal spheroids. Previous findings highlighted the potent efficacy of intratumoral CPMV against OvCa in mouse tumor models. Leveraging the precision control over material deposition and cell patterning afforded by digital-light-processing (DLP) based bioprinting, we constructed OvCa-macrophage spheroids to mimic peritoneal spheroids using gelatin methacrylate (GelMA), a collagen-derived photopolymerizable biomaterial to mimic the extracellular matrix. Following CPMV treatment, bioprinted spheroids exhibited inhibited OvCa progression mediated by macrophage activation. Our analysis indicates that CPMV regulates and activates macrophage to both induce OvCa cell killing and restore normal cell-cell junctions. This study deepened our understanding of the mechanism of CPMV intratumoral immunotherapy in the setting of OvCa. This study also highlights the potential of studying immunotherapies using high throughput tissue models via DLP bioprinting.

High-throughput organo-on-pillar (high-TOP) array system for three-dimensional ex vivo drug testing

The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy. The three-dimensional culture encapsulated with basement membrane (BM) components is extremely important in establishing ex vivo organoids and drug sensitivity tests because the BM components confer essential structures resembling tumor histopathology. Although numerous studies have demonstrated three-dimensional culture-based drug screening methods, establishing a large-scale drug-screening platform with matrix-encapsulated tumor cells is challenging because the arrangement of microspots of a matrix-cell droplet onto each well of a microwell plate is inconsistent and difficult to standardize. In addition, relatively low scales and lack of reproducibility discourage the application of three-dimensional organoid-based drug screening data for precision treatment or drug discovery. To overcome these limitations, we manufactured an automated organospotter-integrated high-throughput organo-on-pillar (high-TOP) drug-screening platform. Our system is compatible with various extracellular matrices, including BM extract, Matrigel, collagen, and hydrogel. In addition, it can be readily utilized for high-content analyses by simply exchanging the bottom plates without disrupting the domes. Our system demonstrated considerable robustness, consistency, reproducibility, and biological relevancy in three-dimensional drug sensitivity analyses using Matrigel-encapsulated ovarian cancer cell lines. We also demonstrated proof-of-concept cases representing the clinical feasibility of high-TOP-assisted ex vivo drug tests linked to clinical chemo-response in ovarian cancer patients. In conclusion, our platform provides an automated and standardized method for ex vivo drug-sensitivity-guided clinical response prediction, suggesting effective chemotherapy regimens for patients with cancer.

Photosensitizer-induced HPV16 E7 nanovaccines for cervical cancer immunotherapy

There is a lack of effective treatment methods for advanced cervical, and the therapeutic HPV vaccine is a promising option. The design of an efficient therapeutic vaccine is one of the main challenges. In the current study, we constructed a novel HPV nanovaccine that could effectively inhibit the progression of cervical cancer by combining nanotechnology and photodynamic therapy. This nanovaccine was constructed by linking bovine serum albumin (BSA) with the E7 antigen and then encapsulating the photosensitizer and adjuvant through disulfide bonds to form a highly biocompatible and stable structure. Due to its slow-release properties and targeted delivery to lymph nodes combined with infrared laser irradiation of photosensitizers to produce a photo-oxygen response, this vaccine could effectively induce the maturation of dendritic cells and stimulate T cell effects, thereby enhancing antitumor immunity. This prevention and treatment of tumors was experimentally demonstrated in a TC-1 cervical cancer model in C57BL/6 mice. This strategy can be applied to the design of therapeutic HPV vaccines in the future for clinical use.

Phototheranostics: An advanced approach for precise diagnosis and treatment of gynecological inflammation and tumors

Fully synthetic nanofibrillar hydrogel for the growth and enzyme-free release of patient-derived ovarian tumor organoids

Patient-derived tumor organoids (PTOs) closely recapitulate the histopathology, gene expression, and drug response of patient tumor tissues, thereby making them highly reliable in vitro models in preclinical cancer research. Currently, basement membrane extracts (BME or Matrigel) dominate the in vitro culture of PTO models, although BME hydrogels suffer from batch-to-batch variability, possible contamination of murine host cells, and non-tunable mechanical properties. Here, we design and prepare fully synthetic thermoreversible nanofibrillar hydrogels (TNHs) for the growth and enzyme-free release of ovarian PTOs. PTOs cultured in TNHs exhibit morphological, histological, and gene expression similarities to organoids grown in Matrigel and their corresponding tumor tissues. In addition, the on-demand release of PTOs without loss of cell viability and organoid integrity is achieved upon cooling. The released PTOs show a strong tumorigenic ability by xenografts of PTOs in mice. The results show that the fully synthetic TNHs is a highly promising alternative to BME for the establishment of ovarian PTOs in vitro.

Amplification of cGAS-STING pathway with “single-molecule multitarget” nanoparticles for chemo-immunotherapy of ovarian cancer

With a low autoimmune risk and dedicated induction of type Ⅰ interferon production in immunotherapy, "STING therapy" holds broad prospects in the treatment of aggressive and metastatic cancers such as ovarian cancer (OC). Inducing pyroptosis constitutes a promising approach for activating the anti-tumor immune response. Nevertheless, compared to combination therapies, "single-molecule multitarget" drugs possess the merits of a lower interaction risk, more predictable pharmacokinetics and a lower cost of clinical trials. Therefore, the present study was conducted to construct a structurally stable nanoparticles (TPAQu-Pt@HA NPs) with controlled drug release behavior and active targeting ability based on a self-designed and synthesized photo-platinum compound (TPAQu-Pt) with aggregation-induced emission (AIE) effect without excipients. The NPs attained "single-molecule multitarget" effect via three mechanisms: 1) causing nuclear and mitochondrial DNA damage and cytoplasmic leakage of double-stranded DNA (dsDNA), effectively activating cGAS-STING pathway; 2) inducing pyroptosis benefited from the AIE effect conferring a stronger ROS-generating capacity; 3) photothermal therapy worsened mitochondrial dysfunction and intensified pyroptosis, amplifying activation of cGAS-STING pathway and the subsequent anti-tumor immune response. In conclusion, this study provided a scientific basis for the molecular modification of cisplatin, which was expected to improve the treatment status of OC.

Trojan-horse inspired nanoblaster: X-ray triggered spot attack on radio-resistant cancer through radiodynamic therapy

Radiotherapy as a mainstay of in-depth cervical cancer (CC) treatment suffers from its radioresistance. Radiodynamic therapy (RDT) effectively reverses radio-resistance by generating reactive oxygen species (ROS) with deep tissue penetration. However, the photosensitizers stimulated by X-ray have high toxicity and energy attenuation. Therefore, X-ray responsive diselenide-bridged mesoporous silica nanoparticles (DMSNs) are designed, loading X-ray-activated photosensitizer acridine orange (AO) for spot blasting RDT like Trojan-horse against radio-resistance cervical cancer (R-CC). DMSNs can encapsulate a large amount of AO, in the tumor microenvironment (TME), which has a high concentration of hydrogen peroxide, X-ray radiation triggers the cleavage of diselenide bonds, leading to the degradation of DMSNs and the consequent release of AO directly at the tumor site. On the one hand, it solves the problems of rapid drug clearance, adverse distribution, and side effects caused by simple AO treatment. On the other hand, it fully utilizes the advantages of highly penetrating X-ray responsive RDT to enhance radiotherapy sensitivity. This approach results in ROS-induced mitochondria damage, inhibition of DNA damage repair, cell cycle arrest and promotion of cancer cell apoptosis in R-CC. The X-ray responsive DMSNs@AO hold considerable potential in overcoming obstacles for advanced RDT in the treatment of R-CC.

A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis

Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.

Selenium promotes immunogenic radiotherapy against cervical cancer metastasis through evoking P53 activation

Radiotherapy is still the recommended treatment for cervical cancer. However, radioresistance and radiation-induced side effects remain one of the biggest clinical problems. Selenium (Se) has been confirmed to exhibit radiation-enhancing effects for cancer treatment. However, Se species dominate the biological activities and which form of Se possesses better radiosensitizing properties and radiation safety remains elusive. Here, different Se species (the valence state of Se ranged from - 2, 0, +4 to + 6) synergy screen was carried out to identify the potential radiosensitizing effects and radiation safety of Se against cervical cancer. We found that the therapeutic effects varied with the changes in the Se valence state. Sodium selenite (+4) displayed strong cancer-killing effects but also possessed severe cytotoxicity. Sodium selenate (+6) neither enhanced the killing effects of X-ray nor possessed anticancer activity by its alone treatment. Although nano-selenium (0), especially Let-SeNPs, has better radiosensitizing activity, the - 2 organic Se, such as selenadiazole derivative SeD (-2) exhibited more potent anticancer effects and possessed a higher safe index. Overall, the selected Se drugs were able to synergize with X-ray to inhibit cell growth, clone formation, and cell migration by triggering G2/M phase arrest and apoptosis, and SeD (-2) was found to exhibit more potent enhancing capacity. Further mechanism studies showed that SeD mediated p53 pathway activation by inducing DNA damage through promoting ROS production. Additionally, SeD combined with X-ray therapy can induce an anti-tumor immune response in vivo. More importantly, SeD combined with X-ray significantly inhibited the liver metastasis of tumor cells and alleviated the side effects caused by radiation therapy in tumor-bearing mice. Taken together, this study demonstrates the radiosensitization and radiation safety effects of different Se species, which may shed light on the application of such Se-containing drugs serving as side effects-reducing agents for cervical cancer radiation treatment.