Bioinspired 3D Culture in Nanoliter Hyaluronic Acid-Rich Core-Shell Hydrogel Microcapsules Isolates Highly Pluripotent Human iPSCs.

Human induced pluripotent stem cells (iPSCs) are ideal for developing personalized medicine. However, the spontaneous differentiation of human iPSCs under conventional 2D and 3D cultures results in significant heterogeneity and compromised quality. Therefore, a method for effectively isolating and expanding high-quality human iPSCs is critically needed. Here, a biomimetic microencapsulation approach for isolating and culturing high-quality human iPSCs is reported. This is inspired by the natural proliferation and development of blastomeres into early blastocyst where the early embryonic stem cells-containing core is enclosed in a semipermeable hydrogel shell known as the zona pellucida (Zona). Blastomere cluster-like human iPSC clusters are encapsulated in a miniaturized (≈10 nanoliter) hyaluronic acid (HA)-rich core of microcapsules with a semipermeable Zona-like hydrogel shell and subsequently cultured to form pluripotent human iPSC spheroids with significantly improved quality. This is indicated by their high expression of pluripotency markers and highly efficient 3D cardiac differentiation. In particular, HA is found to be crucial for isolating the high-quality human iPSCs with the biomimetic core-shell microencapsulation culture. Interestingly, the isolated human iPSCs can maintain high pluripotency even after being cultured again in 2D. These discoveries and the bioinspired culture method may be valuable to facilitate the human iPSC-based personalized medicine.

Deep Learning-Enabled Label-Free On-Chip Detection and Selective Extraction of Cell Aggregate-Laden Hydrogel Microcapsules.

Microfluidic encapsulation of cells/tissues in hydrogel microcapsules has attracted tremendous attention in the burgeoning field of cell-based medicine. However, when encapsulating rare cells and tissues (e.g., pancreatic islets and ovarian follicles), the majority of the resultant hydrogel microcapsules are empty and should be excluded from the sample. Furthermore, the cell-laden hydrogel microcapsules are usually suspended in an oil phase after microfluidic generation, while the microencapsulated cells require an aqueous phase for further culture/transplantation and long-term suspension in oil may compromise the cells/tissues. Thus, real-time on-chip selective extraction of cell-laden hydrogel microcapsules from oil into aqueous phase is crucial to the further use of the microencapsulated cells/tissues. Contemporary extraction methods either require labeling of cells for their identification along with an expensive detection system or have a low extraction purity (<≈30%). Here, a deep learning-enabled approach for label-free detection and selective extraction of cell-laden microcapsules with high efficiency of detection (≈100%) and extraction (≈97%), high purity of extraction (≈90%), and high cell viability (>95%) is reported. The utilization of deep learning to dynamically analyze images in real time for label-free detection and on-chip selective extraction of cell-laden hydrogel microcapsules is unique and may be valuable to advance the emerging cell-based medicine.

Preparation of a mitochondria-targeted and NO-releasing nanoplatform and its enhanced pro-apoptotic effect on cancer cells.

The therapeutic applications of exogenous nitric oxide are usually limited by its short half-life and its vulnerability to many biological substances, thus straightforward and precise spatiotemporal control of NO delivery may be critical to its therapeutic effects. Herein, the mitochondria-targeted and photoresponsive NO-releasing nanosystem is demonstrated as a new approach for cancer treatment. The nanosystem is fabricated by covalently incorporating a NO photo-donor and a mitochondria targeting ligand onto carbon-dots; accordingly, multi-functionalities (mitochondria-targeting, light-enhanced efficient NO-releasing, and cell imaging) are achieved. The in vitro NO release profiles for the nanosystem show that the duration of NO release from the present C-dot-based nanosystem containing immobilized SNO can be extended up to 8 hours or more. Upon cellular internalization, the nanosystem can target mitochondria and release NO. The action of the nanosystem on three cancer cell lines is evaluated; it is found that the targeted NO-releasing system can cause high cytotoxicity towards the cancer cells by specifically damaging their mitochondria. Additionally, light irradiation can amplify the cell apoptosis by enhancing NO release. These observations demonstrate that incorporating mitochondria-targeting ligand onto a NO-releasing system can enhance its pro-apoptosis action, thereby providing new insights for exploiting NO in cancer therapy.

Enhanced photodynamic efficiency achieved via a dual-targeted strategy based on photosensitizer/micelle structure.

The applications of photodynamic therapy (PDT) are usually limited by photosensitizer's side effect and the singlet oxygen's short half-life. Herein, we demonstrate a dual-targeting (both cellular and subcellular targeting) strategy to enhance the PDT efficacy. A cationic porphyrin derivative (MitoTPP) was synthesized as the mitochondrion-targeting photosensitizer, and the dual-targeting PDT system was then fabricated by encapsulating MitoTPP into the acid-responsive and folic acid (FA)-modified polymer micelles. Under acidic pH, the micelles swell as a result of protonation of tertiary amines and disruption of the nucleobase pairing, thereby causing the release of the photosensitizer. Confocal microscope observation shows that the dual-targeting and micelle-based PDT system can preferably enter folate receptor (FR)-positive cancer cells, and upon cellular internalization, the MitoTPP molecules are released from the micelles and selectively accumulate in mitochondria. Under light irradiation, the singlet oxygen generated by the photosensitizer causes the oxidant damage to the mitochondrial and subsequently the apoptosis of the cells, as evidenced by the loss of mitochondrial membrane potential. Cell viability assays indicate that dual-targeting micelle-based systems exhibit enhanced cytotoxicity toward FR-positive cells. This study may provide a new approach for effectively enhancing the action of PDT systems.

Design and preclinical evaluation of a universal SARS-CoV-2 mRNA vaccine.

Because of the rapid mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an effective vaccine against SARS-CoV-2 variants is needed to prevent coronavirus disease 2019 (COVID-19). T cells, in addition to neutralizing antibodies, are an important component of naturally acquired protective immunity, and a number of studies have shown that T cells induced by natural infection or vaccination contribute significantly to protection against several viral infections including SARS-CoV-2. However, it has never been tested whether a T cell-inducing vaccine can provide significant protection against SARS-CoV-2 infection in the absence of preexisting antibodies. In this study, we designed and evaluated lipid nanoparticle (LNP) formulated mRNA vaccines that induce only T cell responses or both T cell and neutralizing antibody responses by using two mRNAs. One mRNA encodes SARS-CoV-2 Omicron Spike protein in prefusion conformation for induction of neutralizing antibodies. The other mRNA encodes over one hundred T cell epitopes (multi-T cell epitope or MTE) derived from non-Spike but conserved regions of the SARS-CoV-2. We show immunization with MTE mRNA alone protected mice from lethal challenge with the SARS-CoV-2 Delta variant or a mouse-adapted virus MA30. Immunization with both mRNAs induced the best protection with the lowest viral titer in the lung. These results demonstrate that induction of T cell responses, in the absence of preexisting antibodies, is sufficient to confer protection against severe disease, and that a vaccine containing mRNAs encoding both the Spike and MTE could be further developed as a universal SARS-CoV-2 vaccine.

In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy.

Cancer immunotherapy that deploys the host's immune system to recognize and attack tumors, is a promising strategy for cancer treatment. However, its efficacy is greatly restricted by the immunosuppressive (i.e., immunologically cold) tumor microenvironment (TME). Here, we report an in-situ cryo-immune engineering (ICIE) strategy for turning the TME from immunologically "cold" into "hot". In particular, after the ICIE treatment, the ratio of the CD8+ cytotoxic T cells to the immunosuppressive regulatory T cells is increased by more than 100 times in not only the primary tumors with cryosurgery but also distant tumors without freezing. This is achieved by combining cryosurgery that causes "frostbite" of tumor with cold-responsive nanoparticles that not only target tumor but also rapidly release both anticancer drug and PD-L1 silencing siRNA specifically into the cytosol upon cryosurgery. This ICIE treatment leads to potent immunogenic cell death, which promotes maturation of dendritic cells and activation of CD8+ cytotoxic T cells as well as memory T cells to kill not only primary but also distant/metastatic breast tumors in female mice (i.e., the abscopal effect). Collectively, ICIE may enable an efficient and durable way to leverage the immune system for combating cancer and its metastasis.

Recent Advancements in Mitochondria-Targeted Nanoparticle Drug Delivery for Cancer Therapy.

Mitochondria are critical subcellular organelles that produce most of the adenosine triphosphate (ATP) as the energy source for most eukaryotic cells. Moreover, recent findings show that mitochondria are not only the "powerhouse" inside cells, but also excellent targets for inducing cell death via apoptosis that is mitochondria-centered. For several decades, cancer nanotherapeutics have been designed to specifically target mitochondria with several targeting moieties, and cause mitochondrial dysfunction via photodynamic, photothermal, or/and chemo therapies. These strategies have been shown to augment the killing of cancer cells in a tumor while reducing damage to its surrounding healthy tissues. Furthermore, mitochondria-targeting nanotechnologies have been demonstrated to be highly efficacious compared to non-mitochondria-targeting platforms both in vitro and in vivo for cancer therapies. Moreover, mitochondria-targeting nanotechnologies have been intelligently designed and tailored to the hypoxic and slightly acidic tumor microenvironment for improved cancer therapies. Collectively, mitochondria-targeting may be a promising strategy for the engineering of nanoparticles for drug delivery to combat cancer.

Greatly Enhanced CTC Culture Enabled by Capturing CTC Heterogeneity Using a PEGylated PDMS-Titanium-Gold Electromicrofluidic Device with Glutathione-Controlled Gentle Cell Release.

The circulating tumor cells (CTCs, the root cause of cancer metastasis and poor cancer prognosis) are very difficult to culture for scale-up in vitro, which has hampered their use in cancer research/prognosis and patient-specific therapeutic development. Herein, we report a robust electromicrofluidic chip for not only efficient capture of heterogeneous (EpCAM+ and CD44+) CTCs with high purity but also glutathione-controlled gentle release of the CTCs with high efficiency and viability. This is enabled by coating the polydimethylsiloxane (PDMS) surface in the device with a 10 nm gold layer through a 4 nm titanium coupling layer, for convenient PEGylation and linkage of capture antibodies via the thiol-gold chemistry. Surprisingly, the percentage of EpCAM+ mammary CTCs can be as low as ∼35% (∼70% on average), showing that the commonly used approach of capturing CTCs with EpCAM alone may miss many EpCAM- CTCs. Furthermore, the CD44+ CTCs can be cultured to form 3D spheroids efficiently for scale-up. In contrast, the CTCs captured with EpCAM alone are poor in proliferation in vitro, consistent with the literature. By capture of the CTC heterogeneity, the percentage of stage IV patients whose CTCs can be successfully cultured/scaled up is improved from 12.5% to 68.8%. These findings demonstrate that the common practice of CTC capture with EpCAM alone misses the CTC heterogeneity including the critical CD44+ CTCs. This study may be valuable to the procurement and scale-up of heterogeneous CTCs, to facilitate the understanding of cancer metastasis and the development of cancer metastasis-targeted personalized cancer therapies conveniently via the minimally invasive liquid/blood biopsy.

Metformin Bicarbonate-Mediated Efficient RNAi for Precise Targeting of TP53 Deficiency in Colon and Rectal Cancers.

Colon and rectal cancers are the leading causes of cancer-related deaths in the United States and effective targeted therapies are in need for treating them. Our genomic analyses show hemizygous deletion of TP53, an important tumor suppressor gene, is highly frequent in both cancers, and the 5-year survival of patients with the more prevalent colon cancer is significantly reduced in the patients with the cancer harboring such deletion, although such reduction is not observed for rectal cancer. Unfortunately, direct targeting TP53 has been unsuccessful for cancer therapy. Interestingly, POLR2A, a gene essential for cell survival and proliferation, is almost always deleted together with TP53 in colon and rectal cancers. Therefore, RNA interference (RNAi) with small interfering RNAs (siRNAs) to precisely target/inhibit POLR2A may be an effective strategy for selectively killing cancer cells with TP53 deficiency. However, the difficulty of delivering siRNAs specifically into the cytosol where they perform their function, is a major barrier for siRNA-based therapies. Here, metformin bicarbonate (MetC) is synthesized to develop pH-responsive MetC-nanoparticles with a unique "bomb" for effective cytosolic delivery of POLR2A siRNA, which greatly facilitates its endo/lysosomal escape into the cytosol and augments its therapeutic efficacy of cancer harboring TP53 deficiency. Moreover, the MetC-based nanoparticles without functional siRNA show notable therapeutic effect with no evident toxicity or immunogenicity.

Carbon nano-onion-mediated dual targeting of P-selectin and P-glycoprotein to overcome cancer drug resistance.

The transmembrane P-glycoprotein (P-gp) pumps that efflux drugs are a major mechanism of cancer drug resistance. They are also important in protecting normal tissue cells from poisonous xenobiotics and endogenous metabolites. Here, we report a fucoidan-decorated silica-carbon nano-onion (FSCNO) hybrid nanoparticle that targets tumor vasculature to specifically release P-gp inhibitor and anticancer drug into tumor cells. The tumor vasculature targeting capability of the nanoparticle is demonstrated using multiple models. Moreover, we reveal the superior light absorption property of nano-onion in the near infrared region (NIR), which enables triggered drug release from the nanoparticle at a low NIR power. The released inhibitor selectively binds to P-gp pumps and disables their function, which improves the bioavailability of anticancer drug inside the cells. Furthermore, free P-gp inhibitor significantly increases the systemic toxicity of a chemotherapy drug, which can be resolved by delivering them with FSCNO nanoparticles in combination with a short low-power NIR laser irradiation.

Engineering Strategies to Improve Islet Transplantation for Type 1 Diabetes Therapy.

Type 1 diabetes is an autoimmune disease in which the immune system attacks insulin-producing beta cells of pancreatic islets. Type 1 diabetes can be treated with islet transplantation; however, patients must be administered immunosuppressants to prevent immune rejection of the transplanted islets if they are not autologous or not engineered with immune protection/isolation. To overcome biological barriers of islet transplantation, encapsulation strategies have been developed and robustly investigated. While islet encapsulation can prevent the need for immunosuppressants, these approaches have not shown much success in clinical trials due to a lack of long-term insulin production. Multiple engineering strategies have been used to improve encapsulation and post-transplantation islet survival. In addition, more efficient islet cryopreservation methods have been designed to facilitate the scaling-up of islet transplantation. Other islet sources have been identified including porcine islets and stem cell-derived islet-like aggregates. Overall, islet-laden capsule transplantation has greatly improved over the past 30 years and is moving towards becoming a clinically feasible treatment for type 1 diabetes.

Targeted Heating of Mitochondria Greatly Augments Nanoparticle-Mediated Cancer Chemotherapy.

Cancer is the second leading cause of mortality globally. Various nanoparticles have been developed to improve the efficacy and safety of chemotherapy, photothermal therapy, and their combination for treating cancer. However, most of the existing nanoparticles are low in both subcellular precision and drug loading content (<≈5%), and the effect of targeted heating of subcellular organelles on the enhancement of chemotherapy has not been well explored. Here, a hybrid Py@Si-TH nanoparticle is reported to first target cancer cells overexpressed with the variant CD44 via its natural ligand HA on the outermost surface of the nanoparticle before cellular uptake, and then target mitochondria after they are taken up inside cells. In addition, the nanoparticle is ultraefficient for encapsulating doxorubicin hydrochloride (DOX) to form Py@Si-TH-DOX nanoparticle. The encapsulation efficiency is ≈100% at the commonly used low feeding ratio of 1:20 (DOX:empty nanoparticle), and >80% at an ultrahigh feeding ratio of 1:1. In combination with near infrared (NIR, 808 nm) laser irradiation, the tumor weight in the Py@Si-TH-DOX treatment group is 8.5 times less than that in the Py@Si-H-DOX (i.e., DOX-laden nanoparticles without mitochondrial targeting) group, suggesting targeted heating of mitochondria is a valuable strategy for enhancing chemotherapy to combat cancer.

Author Correction: Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Creating a capture zone in microfluidic flow greatly enhances the throughput and efficiency of cancer detection.

Efficient capture of rare circulating tumor cells (CTCs) from blood samples is valuable for early cancer detection to improve the management of cancer. In this work, we developed a highly efficient microfluidics-based method for detecting CTCs in human blood. This is achieved by creating separate capture and flow zones in the microfluidic device (ZonesChip) and using patterned dielectrophoretic force to direct cells from the flow zone into the capture zone. This separation of the capture and flow zones minimizes the negative impact of high flow speed (and thus high throughput) and force in the flow zone on the capture efficiency, overcoming a major bottleneck of contemporary microfluidic approaches using overlapping flow and capture zones for CTC detection. When the flow speed is high (≥0.58 mm/s) in the flow zone, the separation of capture and flow zones in our ZonesChip could improve the capture efficiency from ∼0% (for conventional device without separating the two zones) to ∼100%. Our ZonesChip shows great promise as an effective platform for the detection of CTCs in blood from patients with early/localized-stage colorectal tumors.

Publisher Correction: Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer.

The Supplementary Information originally published with this Article was an older version, in which 'IFN-γ' was misspelled 'INF-γ' in Supplementary Fig. 9 and the ß-Actin blot in Supplementary Fig. 13 was the wrong image. The Supplementary Information has now been replaced.

Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer.

TP53 is the most frequently mutated or deleted gene in triple negative breast cancer (TNBC). Both the loss of TP53 and the lack of targeted therapy are significantly correlated with poor clinical outcomes, making TNBC the only type of breast cancer that has no approved targeted therapies. Through in silico analysis, we identified POLR2A in the TP53-neighbouring region as a collateral vulnerability target in TNBC tumours, suggesting that its inhibition via small interfering RNA (siRNA) may be an amenable approach for TNBC targeted treatment. To enhance bioavailability and improve endo/lysosomal escape of siRNA, we designed pH-activated nanoparticles for augmented cytosolic delivery of POLR2A siRNA (siPol2). Suppression of POLR2A expression with the siPol2-laden nanoparticles leads to enhanced growth reduction of tumours characterized by hemizygous POLR2A loss. These results demonstrate the potential of the pH-responsive nanoparticle and the precise POLR2A targeted therapy in TNBC harbouring the common TP53 genomic alteration.

Enhanced cancer therapy with cold-controlled drug release and photothermal warming enabled by one nanoplatform.

Stimuli-responsive nanoparticles hold great promise for drug delivery to improve the safety and efficacy of cancer therapy. One of the most investigated stimuli-responsive strategies is to induce drug release by heating with laser, ultrasound, or electromagnetic field. More recently, cryosurgery (also called cryotherapy and cryoablation), destruction of diseased tissues by first cooling/freezing and then warming back, has been used to treat various diseases including cancer in the clinic. Here we developed a cold-responsive nanoparticle for controlled drug release as a result of the irreversible disassembly of the nanoparticle when cooled to below ∼10 °C. Furthermore, this nanoparticle can be used to generate localized heating under near infrared (NIR) laser irradiation, which can facilitate the warming process after cooling/freezing during cryosurgery. Indeed, the combination of this cold-responsive nanoparticle with ice cooling and NIR laser irradiation can greatly augment cancer destruction both in vitro and in vivo with no evident systemic toxicity.

Targeting 17q23 amplicon to overcome the resistance to anti-HER2 therapy in HER2+ breast cancer.

Chromosome 17q23 amplification occurs in ~11% of human breast cancers. Enriched in HER2+ breast cancers, the 17q23 amplification is significantly correlated with poor clinical outcomes. In addition to the previously identified oncogene WIP1, we uncover an oncogenic microRNA gene, MIR21, in a majority of the WIP1-containing 17q23 amplicons. The 17q23 amplification results in aberrant expression of WIP1 and miR-21, which not only promotes breast tumorigenesis, but also leads to resistance to anti-HER2 therapies. Inhibiting WIP1 and miR-21 selectively inhibits the proliferation, survival and tumorigenic potential of the HER2+ breast cancer cells harboring 17q23 amplification. To overcome the resistance of trastuzumab-based therapies in vivo, we develop pH-sensitive nanoparticles for specific co-delivery of the WIP1 and miR-21 inhibitors into HER2+ breast tumors, leading to a profound reduction of tumor growth. These results demonstrate the great potential of the combined treatment of WIP1 and miR-21 inhibitors for the trastuzumab-resistant HER2+ breast cancers.

Predehydration and Ice Seeding in the Presence of Trehalose Enable Cell Cryopreservation.

Conventional approaches for cell cryopreservation require the use of toxic membrane-penetrating cryoprotective agents (pCPA), which limits the clinical application of cryopreserved cells. Here, we show intentionally induced ice formation at a high subzero temperature (> -10 °C) during cryopreservation, which is often referred to as ice seeding, could result in significant cell injury in the absence of any pCPA. This issue can be mitigated by predehydrating cells using extracellular trehalose to their minimal volume with minimized osmotically active water before ice seeding. We further observe that ice seeding can minimize the interfacial free energy that drives the devastating ice recrystallization-induced cell injury during warming cryopreserved samples. Indeed, by combining predehydration using extracellular trehalose with ice seeding at high subzero temperatures, high cell viability or recovery is achieved for fibroblasts, adult stem cells, and red blood cells after cryopreservation without using any pCPA. The pCPA-free technology developed in this study may greatly facilitate the long-term storage and ready availability of living cells, tissues, and organs that are of high demand by modern cell-based medicine.