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.

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.

Noncovalent reversible binding-enabled facile fabrication of leak-free PDMS microfluidic devices without plasma treatment for convenient cell loading and retrieval.

The conventional approach for fabricating polydimethylsiloxane (PDMS) microfluidic devices is a lengthy and inconvenient procedure and may require a clean-room microfabrication facility often not readily available. Furthermore, living cells can't survive the oxygen-plasma and high-temperature-baking treatments required for covalent bonding to assemble multiple PDMS parts into a leak-free device, and it is difficult to disassemble the devices because of the irreversible covalent bonding. As a result, seeding/loading cells into and retrieving cells from the devices are challenging. Here, we discovered that decreasing the curing agent for crosslinking the PDMS prepolymer increases the noncovalent binding energy of the resultant PDMS surfaces without plasma or any other treatment. This enables convenient fabrication of leak-free microfluidic devices by noncovalent binding for various biomedical applications that require high pressure/flow rates and/or long-term cell culture, by simply hand-pressing the PDMS parts without plasma or any other treatment to bind/assemble. With this method, multiple types of cells can be conveniently loaded into specific areas of the PDMS parts before assembly and due to the reversible nature of the noncovalent bonding, the assembled device can be easily disassembled by hand peeling for retrieving cells. Combining with 3D printers that are widely available for making masters to eliminate the need of photolithography, this facile yet rigorous fabrication approach is much faster and more convenient for making PDMS microfluidic devices than the conventional oxygen plasma-baking-based irreversible covalent bonding method.

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.

Oxidation and RGD Modification Affect the Early Neural Differentiation of Murine Embryonic Stem Cells Cultured in Core-Shell Alginate Hydrogel Microcapsules.

Directed neural differentiation of embryonic stem cells (ESCs) has been studied extensively to improve the treatment of neurodegenerative disorders. This can be done through stromal-cell derived inducing activity (SDIA), by culturing ESCs directly on top of a layer of feeder stromal cells. However, the stem cells usually become mixed with the feeder cells during the differentiation process, making it difficult to obtain a pure population of the differentiated cells for further use. To address this issue, a non-planar microfluidic device is used here to encapsulate murine ESCs (mESCs) in the 3D liquid core of microcapsules with an alginate hydrogel shell of different sizes for early neural differentiation through SDIA, by culturing mESC-laden microcapsules over a feeder layer of PA6 cells. Furthermore, the alginate hydrogel shell of the microcapsules is modified via oxidation or RGD peptide conjugation to examine the mechanical and chemical effects on neural differentiation of the encapsulated mESC aggregates. A higher expression of Nestin is observed in the aggregates encapsulated in small (∼300 µm) microcapsules and cultured over the PA6 cell feeder layer. Furthermore, the modification of the alginate with RGD facilitates early neurite extension within the microcapsules. This study demonstrates that the presence of the RGD peptide, the SDIA effect of the PA6 cells, and the absence of leukemia inhibition factor from the medium can lead to the early differentiation of mESCs with extensive neurites within the 3D microenvironment of the small microcapsules. This is the first study to investigate the effects of cell adhesion and degradation of the encapsulation materials for directed neural differentiation of mESCs. The simple modifications (i.e., oxidation and RGD incorporation) of the miniaturized 3D environment for improved early neural differentiation of mESCs may potentially enhance further downstream differentiation of the mESCs into more specialized neurons for therapeutic use and drug screening.

Rock inhibitor may compromise human induced pluripotent stem cells for cardiac differentiation in 3D.

Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) are valuable for the understanding/treatment of the deadly heart diseases and their drug screening. However, the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging. Here, it is discovered surprisingly that Rock inhibitor (RI), used ubiquitously to improve the survival/yield of human iPSCs, induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 µM). This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 µM for 3D culture, the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95 % in ~7 days (compared to less than ~50 % in 14 days for the 10 µM RI condition). Furthermore, the outset beating time (OBT) of all resultant cardiac spheroids (CSs) is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol (GelMA) hydrogel, showing high homogeneity (in terms of the OBT) in functional maturity of the CSs. Moreover, the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs. These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases.

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.

Image entropy-based label-free functional characterization of human induced pluripotent stem cell-derived 3D cardiac spheroids.

Human induced pluripotent stem cell-derived cardiac spheroids (iPSC-CSs) in 3D possess tremendous potential for treating heart diseases and screening drugs for their cardiac effect. The beating pattern (including beating frequency and amplitude) of iPSC-CSs is a direct indicator of their health and function. However, detecting the beating pattern of 3D cardiac spheroid is not well studied and the probes commonly used for labeling cardiomyocytes for their beating pattern detection is toxic during long-term culture. Here, we reveal that the beating pattern of 3D iPSC-CSs can be conveniently detected/quantified by calculating the relative change of entropy in all the frames/images of non-fluorescent optical signal without labeling any cells. The entropy rate superpixel segmentation method is used for image segmentation in frames containing multiple or aggregated iPSC-CSs to identify individual iPSC-CSs, enabling rapid detection/quantification of the beating pattern of each iPSC-CS. Moreover, the responses of iPSC-CSs to both anticancer and cardiac drugs can be reliably detected with the image entropy-based label-free method in terms of their beating patterns. This novel label-free approach may be valuable for convenient and efficient functional evaluation of 3D and 2D cardiac constructs, which is important not only for drug screening but also the advancement of manufacturing functional cardiac constructs to treat heart diseases.

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.

Stem cell therapy of myocardial infarction: a promising opportunity in bioengineering.

Myocardial infarction (MI) is a life-threatening disease resulting from irreversible death of cardiomyocytes (CMs) and weakening of the heart blood-pumping function. Stem cell-based therapies have been studied for MI treatment over the last two decades with promising outcome. In this review, we critically summarize the past work in this field to elucidate the advantages and disadvantages of treating MI using pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), adult stem cells, and cardiac progenitor cells. The main advantage of the latter is their cytokine production capability to modulate immune responses and control the progression of healing. However, human adult stem cells have very limited (if not 'no') capacity to differentiate into functional CMs in vitro or in vivo. In contrast, PSCs can be differentiated into functional CMs although the protocols for the cardiac differentiation of PSCs are mainly for adherent cells under 2D culture. Derivation of PSC-CMs in 3D, allowing for large-scale production of CMs via modulation of the Wnt/ß-catenin signal pathway with defined chemicals and medium, may be desired for clinical translation. Furthermore, the technology of purification and maturation of the PSC-CMs may need further improvements to eliminate teratoma formation after in vivo implantation of the PSC-CMs for treating MI. In addition, in vitro derived PSC-CMs may have mechanical and electrical mismatch with the patient's cardiac tissue, which causes arrhythmia. This supports the use of PSC-derived cells committed to cardiac lineage without beating for implantation to treat MI. In this case, the PSC derived cells may utilize the mechanical, electrical, and chemical cues in the heart to further differentiate into mature/functional CMs in situ. Another major challenge facing stem cell therapy of MI is the low retention/survival of stem cells or their derivatives (e.g., PSC-CMs) in the heart for MI treatment after injection in vivo. This may be resolved by using biomaterials to engineer stem cells for reduced immunogenicity, immobilization of the cells in the heart, and increased integration with the host cardiac tissue. Biomaterials have also been applied in the derivation of CMs in vitro to increase the efficiency and maturation of differentiation. Collectively, a lot has been learned from the past failure of simply injecting intact stem cells or their derivatives in vivo for treating MI, and bioengineering stem cells with biomaterials is expected to be a valuable strategy for advancing stem cell therapy towards its widespread application for treating MI in the clinic.

Verteporfin-Loaded Polymeric Microparticles for Intratumoral Treatment of Brain Cancer.

Glioblastoma (GBMs) is the most common and aggressive type of primary brain tumor in adults with dismal prognosis despite radical surgical resection coupled with chemo- and radiotherapy. Recent studies have proposed the use of small-molecule inhibitors, including verteporfin (VP), to target oncogenic networks in cancers. Here we report efficient encapsulation of water-insoluble VP in poly(lactic- co-glycolic acid) microparticles (PLGA MP) of ∼1.5 µm in diameter that allows tunable, sustained release. Treatment with naked VP and released VP from PLGA MP decreased cell viability of patient-derived primary GBM cells in vitro by ∼70%. Moreover, naked VP treatment significantly increased radiosensitivity of GBM cells, thereby enhancing overall tumor cell killing ability by nearly 85%. Our in vivo study demonstrated that two intratumoral administrations of sustained slow-releasing VP-loaded PLGA MPs separated by two weeks significantly attenuated tumor growth by ∼67% in tumor volume in a subcutaneous patient-derived GBM xenograft model over 26 d. Additionally, our in vitro data indicate broader utility of VP for treatment for other solid cancers, including chordoma, malignant meningioma, and various noncentral nervous system-derived carcinomas. Collectively, our work suggests that the use of VP-loaded PLGA MP may be an effective local therapeutic strategy for a variety of solid cancers, including unresectable and orphan tumors, which may decrease tumor burden and ultimately improve patient prognosis.

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.

Verteporfin-Loaded Anisotropic Poly(Beta-Amino Ester)-Based Micelles Demonstrate Brain Cancer-Selective Cytotoxicity and Enhanced Pharmacokinetics.

BACKGROUND: Nanomedicine can improve traditional therapies by enhancing the controlled release of drugs at targeted tissues in the body. However, there still exists disease- and therapy-specific barriers that limit the efficacy of such treatments. A major challenge in developing effective therapies for one of the most aggressive brain tumors, glioblastoma (GBM), is affecting brain cancer cells while avoiding damage to the surrounding healthy brain parenchyma. Here, we developed poly(ethylene glycol) (PEG)-poly(beta-amino ester) (PBAE) (PEG-PBAE)-based micelles encapsulating verteporfin (VP) to increase tumor-specific targeting. METHODS: Biodegradable, pH-sensitive micelles of different shapes were synthesized via nanoprecipitation using two different triblock PEG-PBAE-PEG copolymers varying in their relative hydrophobicity. The anti-tumor efficacy of verteporfin loaded in these anisotropic and spherical micelles was evaluated in vitro using patient-derived primary GBM cells. RESULTS: For anisotropic micelles, uptake efficiency was ~100% in GBM cells (GBM1A and JHGBM612) while only 46% in normal human astrocytes (NHA) at 15.6 nM VP (p ≤ 0.0001). Cell killing of GBM1A and JHGBM612 vs NHA was 52% and 77% vs 29%, respectively, at 24 hrs post-treatment of 125 nM VP-encapsulated in anisotropic micelles (p ≤ 0.0001), demonstrating the tumor cell-specific selectivity of VP. Moreover, anisotropic micelles showed an approximately fivefold longer half-life in blood circulation than the analogous spherical micelles in a GBM xenograft model in mice. In this model, micelle accumulation to tumors was significantly greater for anisotropic micelle-treated mice compared to spherical micelle-treated mice at both 8 hrs (~1.8-fold greater, p ≤ 0.001) and 24 hrs (~2.1-fold greater, p ≤ 0.0001). CONCLUSION: Overall, this work highlights the promise of a biodegradable anisotropic micelle system to overcome multiple drug delivery challenges and enhance efficacy and safety for the treatment of brain cancer.

Verteporfin-Loaded Poly(ethylene glycol)-Poly(beta-amino ester)-Poly(ethylene glycol) Triblock Micelles for Cancer Therapy.

Amphiphilic polymers can be used to form micelles to deliver water-insoluble drugs. A biodegradable poly(ethylene glycol) (PEG)-poly(beta-amino ester) (PBAE)-PEG triblock copolymer was developed that is useful for drug delivery. It was shown to successfully encapsulate and pH-dependently release a water-insoluble, small molecule anticancer drug, verteporfin. PEG-PBAE-PEG micelle morphology was also controlled through variations to the hydrophobicity of the central PBAE block of the copolymer in order to evade macrophage uptake. Spherical micelles were 50 nm in diameter, while filamentous micelles were 31 nm in width with an average aspect ratio of 20. When delivered to RAW 264.7 mouse macrophages, filamentous micelles exhibited a 89% drop in cellular uptake percentage and a 5.6-fold drop in normalized geometric mean cellular uptake compared to spherical micelles. This demonstrates the potential of high-aspect-ratio, anisotropically shaped PEG-PBAE-PEG micelles to evade macrophage-mediated clearance. Both spherical and filamentous micelles also showed therapeutic efficacy in human triple-negative breast cancer and small cell lung cancer cells without requiring photodynamic therapy to achieve an anticancer effect. Both spherical and filamentous micelles were more effective in killing lung cancer cells than breast cancer cells at equivalent verteporfin concentrations, while spherical micelles were shown to be more effective than filamentous micelles against both cancer cells. Spherical and filamentous micelles at 5 and 10 µM respective verteporfin concentration resulted in 100% cell killing of lung cancer cells, but both micelles required a higher verteporfin concentration of 20 µM to kill breast cancer cells at the levels of 80% and 50% respectively. This work demonstrates the potential of PEG-PBAE-PEG as a biodegradable, anisotropic drug delivery system as well as the in vitro use of verteporfin-loaded micelles for cancer therapy.