Reducing Cholesterol Level in Live Macrophages Improves Delivery Performance by Enhancing Blood Shear Stress Adaptation.

In recent years, live-cell-based drug delivery systems have gained considerable attention. However, shear stress, which accompanies blood flow, may cause cell death and weaken the delivery performance. In this study, we found that reducing cholesterol in macrophage plasma membranes enhanced their tumor targeting ability by more than 2-fold. Our study demonstrates that the reduced cholesterol level deactivated the mammalian target of rapamycin (mTOR) and consequently promoted the nuclear translocation of transcription factor EB (TFEB), which in turn enhanced the expression of superoxide dismutase (SOD) to reduce reactive oxygen species (ROS) induced by shear stress. A proof-of-concept system using low cholesterol macrophages attached to MXene (e.g., l-RX) was fabricated. In a melanoma mouse model, l-RX and laser irradiation treatments eliminated tumors with no recurrences observed in mice. Therefore, cholesterol reduction is a simple and effective way to enhance the targeting performance of macrophage-based drug delivery systems.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex ßγ-CAT evolved to drive cell vesicular delivery and transport.

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. ßγ-CAT, a complex of pore-forming protein BmALP1 (two ßγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the ßγ-CAT complex and loss of biological activity. Here, we found that the B. maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for ßγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the ßγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of ßγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

Using Decellularized Magnetic Microrobots to Deliver Functional Cells for Cartilage Regeneration.

The use of natural cartilage extracellular matrix (ECM) has gained widespread attention in the field of cartilage tissue engineering. However, current approaches for delivering functional scaffolds for osteoarthritis (OA) therapy rely on knee surgery, which is limited by the narrow and complex structure of the articular cavity and carries the risk of injuring surrounding tissues. This work introduces a novel cell microcarrier, magnetized cartilage ECM-derived scaffolds (M-CEDSs), which are derived from decellularized natural porcine cartilage ECM. Human bone marrow mesenchymal stem cells are selected for their therapeutic potential in OA treatments. Owing to their natural composition, M-CEDSs have a biomechanical environment similar to that of human cartilage and can efficiently load functional cells while maintaining high mobility. The cells are released spontaneously at a target location for at least 20 days. Furthermore, cell-seeded M-CEDSs show better knee joint function recovery than control groups 3 weeks after surgery in preclinical experiments, and ex vivo experiments reveal that M-CEDSs can rapidly aggregate inside tissue samples. This work demonstrates the use of decellularized microrobots for cell delivery and their in vivo therapeutic effects in preclinical tests.

FAP-retargeted Ad5 enables in vivo gene delivery to stromal cells in the tumor microenvironment.

Fibroblast activation protein (FAP) is a cell surface serine protease that is highly expressed on reactive stromal fibroblasts, such as cancer-associated fibroblasts (CAFs), and generally absent in healthy adult tissues. FAP expression in the tumor stroma has been detected in more than 90% of all carcinomas, rendering CAFs excellent target cells for a tumor site-specific adenoviral delivery of cancer therapeutics. Here, we present a tropism-modified human adenovirus 5 (Ad5) vector that targets FAP through trivalent, designed ankyrin repeat protein-based retargeting adapters. We describe the development and validation of these adapters via cell-based screening assays and demonstrate adapter-mediated Ad5 retargeting to FAP+ fibroblasts in vitro and in vivo. We further show efficient in vivo delivery and in situ production of a therapeutic payload by CAFs in the tumor microenvironment (TME), resulting in attenuated tumor growth. We thus propose using our FAP-Ad5 vector to convert CAFs into a "biofactory," secreting encoded cancer therapeutics into the TME to enable a safe and effective cancer treatment.

Functionalization and magnetonavigation of T-lymphocytes functionalized via nanocomposite capsules targeting with electromagnetic tweezers.

Modification of T-lymphocytes, which are capable of paracellular transmigration is a promising trend in modern personalized medicine. However, the delivery of required concentrations of functionalized T-cells to the target tissues remains a problem. We describe a novel method to functionalize T-cells with magnetic nanocapsules and target them with electromagnetic tweezers. T-cells were modified with the following magnetic capsules: Parg/DEX (150 nm), BSA/TA (300 nm), and BSA/TA (500 nm). T-cells were magnetonavigated in a phantom blood vessel capillary in cultural medium and in whole blood. The permeability of tumor tissues to captured T-cells was analyzed by magnetic delivery of modified T-cells to spheroids formed from 4T1 breast cancer cells. The dynamics of T-cell motion under a magnetic field gradient in model environments were analyzed by particle image velocimetry. The magnetic properties of the nanocomposite capsules and magnetic T-cells were measured. The obtained results are promising for biomedical applications in cancer immunotherapy.

Magnetic Microsphere Scaffold-Based Soft Microbots for Targeted Mesenchymal Stem Cell Delivery.

A soft microbot assembled from individual magnetic microsphere scaffold (MMS) beads carrying mesenchymal stem cells (MSC) is navigated under magnetic actuation, where an oscillating field induces mechanical flexion to propel the microbot toward the target site. A seven-bead microbot attained a top translational speed of 205.6 µm s-1 (0.068 body length s-1 ) under 10 mT and 2 Hz field oscillation. The shallow flexion angle (10-24.5°) allows precision movements required to navigate narrow spaces. Upon arrival at the target site, the MMS beads unload their MSC cargo following exposure to a phosphate-buffered saline (PBS) solution, mimicking the extracellular fluid's sodium concentration. The released stem cells have excellent viability and vitality, promoting rapid healing (i.e., 83.2% vs 49%) in a scratch-wound assay. When paired with minimally invasive surgical methods, such as laparoscopy and endoscopic surgery, the microbot can provide precise stem cell delivery to hard-to-reach injury sites in the body to promote healing. Moreover, the microbot is designed to be highly versatile, with individual MMS beads customizable for cargoes of live cells, biomolecules, bionanomaterials, and pharmaceutical compounds for various therapeutic requirements.

Real-time trajectory guide tracking for intraoperative MRI-guided neurosurgery.

PURPOSE: In current intraoperative MRI (IMRI) methods, an iterative approach is used to aim trajectory guides at intracerebral targets: image MR-visible features, determine current aim by fitting model to image, manipulate device, repeat. Infrequent updates are produced by such methods, compared to rapid optically tracked stereotaxy used in the operating room. Our goal was to develop a real-time interactive IMRI method for aiming. METHODS: The current trajectory was computed from two points along the guide's central axis, rather than by imaging the entire device. These points were determined by correlating one-dimensional spokes from a radial sequence with the known cross-sectional projection of the guide. The real-time platform RTHawk was utilized to control MR sequences and data acquisition. On-screen updates were viewed by the operator while simultaneously manipulating the guide to align it with the planned trajectory. Accuracy was quantitated in a phantom, and in vivo validation was demonstrated in nonhuman primates undergoing preclinical gene ( n = 5 $$ n=5 $$ ) and cell ( n = 4 $$ n=4 $$ ) delivery surgeries. RESULTS: Updates were produced at 5 Hz In 10 phantom experiments at a depth of 48 mm, the cannula tip was placed with radial error of (min, mean, max) = (0.16, 0.29, 0.68) mm. Successful in vivo delivery of payloads to all 14 targets was demonstrated across nine surgeries with depths of (min, mean, max) = (33.3, 37.9, 42.5) mm. CONCLUSION: A real-time interactive update rate was achieved, reducing operator fatigue without compromising accuracy. Qualitative interpretation of images during aiming was rendered unnecessary by objectively computing device alignment.

Improved magnetic delivery of cells to the trabecular meshwork in mice.

Glaucoma is the leading cause of irreversible blindness worldwide and its most prevalent subtype is primary open angle glaucoma (POAG). One pathological change in POAG is loss of cells in the trabecular meshwork (TM), which is thought to contribute to ocular hypertension and has thus motivated development of cell-based therapies to refunctionalize the TM. TM cell therapy has shown promise in intraocular pressure (IOP) control, but existing cell delivery techniques suffer from poor delivery efficiency. We employed a novel magnetic delivery technique to reduce the unwanted side effects of off-target cell delivery. Mesenchymal stem cells (MSCs) were labeled with superparamagnetic iron oxide nanoparticles (SPIONs) and after intracameral injection were magnetically steered towards the TM using a focused magnetic apparatus ("point magnet"). This technique delivered the cells significantly closer to the TM at higher quantities and with more circumferential uniformity compared to either unlabeled cells or those delivered using a "ring magnet" technique. We conclude that our point magnet cell delivery technique can improve the efficiency of TM cell therapy and in doing so, potentially increase the therapeutic benefits and lower the risk of complications such as tumorigenicity and immunogenicity.

Cell delivery systems: Toward the next generation of cell therapies for type 1 diabetes.

Immunoprotection and oxygen supply are vital in implementing a cell therapy for type 1 diabetes (T1D). Without these features, the transplanted islet cell clusters will be rejected by the host immune system, and necrosis will occur due to hypoxia. The use of anti-rejection drugs can help protect the transplanted cells from the immune system; yet, they also may have severe side effects. Cell delivery systems (CDS) have been developed for islet transplantation to avoid using immunosuppressants. CDS provide physical barriers to reduce the immune response and chemical coatings to reduce host fibrotic reaction. In some CDS, there is architecture to support vascularization, which enhances oxygen exchange. In this review, we discuss the current clinical and preclinical studies using CDS without immunosuppression as a cell therapy for T1D. We find that though CDS have been demonstrated for their ability to support immunoisolation of the grafted cells, their functionality has not been fully optimized. Current advanced methods in clinical trials demonstrate the systems are partly functional, physically complicated to implement or inefficient. However, modifications are being made to overcome these issues.

Biostimulatory Micro-Fragmented Nanofiber-Hydrogel Composite Improves Mesenchymal Stem Cell Delivery and Soft Tissue Remodeling.

Functional microgels are preferred stem cell carriers due to the ease of delivery through minimally invasive injection and seamless integration with the surrounding host tissue. A biostimulatory nanofiber-hydrogel composite (NHC) has been previously developed through covalently crosslinking a hyaluronic acid hydrogel network with surface-functionalized poly (ε-caprolactone) nanofiber fragments. The NHC mimics the microarchitecture of native soft tissue matrix, showing enhanced cell infiltration, immunomodulation, and proangiogenic properties. Here, injectability of the pre-formed NHC is improved by mechanical fragmentation, making it into micro-fragmented NHC (mfNHC) in a granular gel form as a stem cell carrier to deliver mesenchymal stem cells (MSCs) for soft tissue remodeling. The mfNHC shows a similar storage modulus but a significantly reduced injection force, as compared with the corresponding bulk NHC. When injected subcutaneously in a rat model, mfNHC-MSC constructs initiate an elevated level of host macrophage infiltration, more pro-regenerative polarization, and subsequently, improved angiogenesis and adipogenesis response when compared to mfNHC alone. A similar trend of host cell infiltration and pro-angiogenic response is detected in a swine model with a larger volume injection. These results suggest a strong potential for use of the mfNHC as an injectable carrier for cell delivery and soft tissue remodeling.

A Biodegradable Magnetic Microrobot Based on Gelatin Methacrylate for Precise Delivery of Stem Cells with Mass Production Capability.

A great deal of research has focused on small-scale robots for biomedical applications and minimally invasive delivery of therapeutics (e.g., cells, drugs, and genes) to a target area. Conventional fabrication methods, such as two-photon polymerization, can be used to build sophisticated micro- and nanorobots, but the long fabrication cycle for a single microrobot has limited its practical use. This study proposes a biodegradable spherical gelatin methacrylate (GelMA) microrobot for mass production in a microfluidic channel. The proposed microrobot is fabricated in a flow-focusing droplet generator by shearing a mixture of GelMA, photoinitiator, and superparamagnetic iron oxide nanoparticles (SPIONs) with a mixture of oil and surfactant. Human nasal turbinate stem cells (hNTSCs) are loaded on the GelMA microrobot, and the hNTSC-loaded microrobot shows precise rolling motion in response to an external rotating magnetic field. The microrobot is enzymatically degraded by collagenase, and released hNTSCs are proliferated and differentiated into neuronal cells. In addition, the feasibility of the GelMA microrobot as a cell therapeutic delivery system is investigated by measuring electrophysiological activity on a multielectrode array. Such a versatile and fully biodegradable microrobot has the potential for targeted stem cell delivery, proliferation, and differentiation for stem cell-based therapy.

Optimal Intravenous Administration Procedure for Efficient Delivery of Canine Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are currently being investigated for their therapeutic applications in a wide range of diseases. Although many studies examined peripheral venous administration of MSC, few have investigated the detailed intravenous administration procedures of MSC from their preparation until they enter the body. The current study therefore aimed to explore the most efficient infusion procedure for MSC delivery by preparing and infusing them under various conditions. Canine adipose-derived mesenchymal stem cells (cADSC) were infused using different infusion apparatuses, suspension solutions, allogenic serum supplementation, infusion time and rates, and cell densities, respectively. Live and dead cell counts were then assessed by manual measurements and flow cytometry. Efficiency of live- and dead-cell infusion and cell viability were calculated from the measured cell counts and compared under each condition. Efficiency of live-cell infusion differed significantly according to the infusion apparatus, infusion rate, and combination of cell density and serum supplementation. Cell viability after infusion differed significantly between the infusion apparatuses. The optimal infusion procedure resulting in the highest cell delivery and viability involved suspending cADSC in normal saline supplemented with 5% allogenic serum at a density of 5 × 105 cells/mL, and infusing them using an automatic infusion device for 15 min. This procedure is therefore recommended as the standard procedure for the intravenous administration of ADSC in terms of cell-delivery efficiency.

Prospects and Challenges of Electrospun Cell and Drug Delivery Vehicles to Correct Urethral Stricture.

Current therapeutic modalities to treat urethral strictures are associated with several challenges and shortcomings. Therefore, significant strides have been made to develop strategies with minimal side effects and the highest therapeutic potential. In this framework, electrospun scaffolds incorporated with various cells or bioactive agents have provided promising vistas to repair urethral defects. Due to the biomimetic nature of these constructs, they can efficiently mimic the native cells' niches and provide essential microenvironmental cues for the safe transplantation of multiple cell types. Furthermore, these scaffolds are versatile platforms for delivering various drug molecules, growth factors, and nucleic acids. This review discusses the recent progress, applications, and challenges of electrospun scaffolds to deliver cells or bioactive agents during the urethral defect repair process. First, the current status of electrospinning in urethral tissue engineering is presented. Then, the principles of electrospinning in drug and cell delivery applications are reviewed. Finally, the recent preclinical studies are summarized and the current challenges are discussed.

Understanding and leveraging cell metabolism to enhance mesenchymal stem cell transplantation survival in tissue engineering and regenerative medicine applications.

In tissue engineering and regenerative medicine, stem cell-specifically, mesenchymal stromal/stem cells (MSCs)-therapies have fallen short of their initial promise and hype. The observed marginal, to no benefit, success in several applications has been attributed primarily to poor cell survival and engraftment at transplantation sites. MSCs have a metabolism that is flexible enough to enable them to fulfill their various cellular functions and remarkably sensitive to different cellular and environmental cues. At the transplantation sites, MSCs experience hostile environments devoid or, at the very least, severely depleted of oxygen and nutrients. The impact of this particular setting on MSC metabolism ultimately affects their survival and function. In order to develop the next generation of cell-delivery materials and methods, scientists must have a better understanding of the metabolic switches MSCs experience upon transplantation. By designing treatment strategies with cell metabolism in mind, scientists may improve survival and the overall therapeutic potential of MSCs. Here, we provide a comprehensive review of plausible metabolic switches in response to implantation and of the various strategies currently used to leverage MSC metabolism to improve stem cell-based therapeutics.

Repair of Retinal Degeneration following Ex Vivo Minicircle DNA Gene Therapy and Transplantation of Corrected Photoreceptor Progenitors.

The authors describe retinal reconstruction and restoration of visual function in heritably blind mice missing the rhodopsin gene using a novel method of ex vivo gene therapy and cell transplantation. Photoreceptor precursors with the same chromosomal genetic mutation were treated ex vivo using minicircle DNA, a non-viral technique that does not present the packaging limitations of adeno-associated virus (AAV) vectors. Following transplantation, genetically modified cells reconstructed a functional retina and supported vision in blind mice harboring the same founder gene mutation. Gene delivery by minicircles showed comparable long-term efficiency to AAV in delivering the missing gene, representing the first non-viral system for robust treatment of photoreceptors. This important proof-of-concept finding provides an innovative convergence of cell and gene therapies for the treatment of hereditary neurodegenerative disease and may be applied in future studies toward ex vivo correction of patient-specific cells to provide an autologous source of tissue to replace lost photoreceptors in inherited retinal blindness. This is the first report using minicircles in photoreceptor progenitors and the first to transplant corrected photoreceptor precursors to restore vision in blind animals.

Efficient and Innocuous Live-Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry: How Better Cellular Delivery Tools Can Contribute to Precise and Quantitative Cell Biology Assays.

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell-based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.

Employing Extracellular Matrix-Based Tissue Engineering Strategies for Age-Dependent Tissue Degenerations.

Tissues and organs are not composed of solely cellular components; instead, they converge with an extracellular matrix (ECM). The composition and function of the ECM differ depending on tissue types. The ECM provides a microenvironment that is essential for cellular functionality and regulation. However, during aging, the ECM undergoes significant changes along with the cellular components. The ECM constituents are over- or down-expressed, degraded, and deformed in senescence cells. ECM aging contributes to tissue dysfunction and failure of stem cell maintenance. Aging is the primary risk factor for prevalent diseases, and ECM aging is directly or indirectly correlated to it. Hence, rejuvenation strategies are necessitated to treat various age-associated symptoms. Recent rejuvenation strategies focus on the ECM as the basic biomaterial for regenerative therapies, such as tissue engineering. Modified and decellularized ECMs can be used to substitute aged ECMs and cell niches for culturing engineered tissues. Various tissue engineering approaches, including three-dimensional bioprinting, enable cell delivery and the fabrication of transplantable engineered tissues by employing ECM-based biomaterials.

Allogenic Use of Human Placenta-Derived Stromal Cells as a Highly Active Subtype of Mesenchymal Stromal Cells for Cell-Based Therapies.

The application of mesenchymal stromal cells (MSCs) from different sources, including bone marrow (BM, bmMSCs), adipose tissue (atMSCs), and human term placenta (hPSCs) has been proposed for various clinical purposes. Accumulated evidence suggests that the activity of the different MSCs is indirect and associated with paracrine release of pro-regenerative and anti-inflammatory factors. A major limitation of bmMSCs-based treatment for autologous application is the limited yield of cells harvested from BM and the invasiveness of the procedure. Similar effects of autologous and allogeneic MSCs isolated from various other tissues were reported. The easily available fresh human placenta seems to represent a preferred source for harvesting abundant numbers of human hPSCs for allogenic use. Cells derived from the neonate tissues of the placenta (f-hPSC) can undergo extended expansion with a low risk of senescence. The low expression of HLA class I and II on f-hPSCs reduces the risk of rejection in allogeneic or xenogeneic applications in normal immunocompetent hosts. The main advantage of hPSCs-based therapies seems to lie in the secretion of a wide range of pro-regenerative and anti-inflammatory factors. This renders hPSCs as a very competent cell for therapy in humans or animal models. This review summarizes the therapeutic potential of allogeneic applications of f-hPSCs, with reference to their indirect pro-regenerative and anti-inflammatory effects and discusses clinical feasibility studies.

Injectable Cardiac Cell Microdroplets for Tissue Regeneration.

One of the strategies for heart regeneration includes cell delivery to the defected heart. However, most of the injected cells do not form quick cell-cell or cell-matrix interactions, therefore, their ability to engraft at the desired site and improve heart function is poor. Here, the use of a microfluidic system is reported for generating personalized hydrogel-based cellular microdroplets for cardiac cell delivery. To evaluate the system's limitations, a mathematical model of oxygen diffusion and consumption within the droplet is developed. Following, the microfluidic system's parameters are optimized and cardiac cells from neonatal rats or induced pluripotent stem cells are encapsulated. The morphology and cardiac specific markers are assessed and cell function within the droplets is analyzed. Finally, the cellular droplets are injected to mouse gastrocnemius muscle to validate cell retention, survival, and maturation within the host tissue. These results demonstrate the potential of this approach to generate personalized cellular microtissues, which can be injected to distinct regions in the body for treating damaged tissues.

Development of Magnet-Driven and Image-Guided Degradable Microrobots for the Precise Delivery of Engineered Stem Cells for Cancer Therapy.

Precise delivery of therapeutic cells to the desired site in vivo is an emerging and promising cellular therapy in precision medicine. This paper presents the development of a magnet-driven and image-guided degradable microrobot that can precisely deliver engineered stem cells for orthotopic liver tumor treatment. The microrobot employs a burr-like porous sphere structure and is made with a synthesized composite to fulfill degradability, mechanical strength, and magnetic actuation capability simultaneously. The cells can be spontaneously released from the microrobots on the basis of the optimized microrobot structure. The microrobot is actuated by a gradient magnetic field and guided by a unique photoacoustic imaging technology. In preclinical experiments on nude mice, microrobots carrying cells are injected via the portal vein and the released cells from the microrobots can inhibit the tumor growth greatly. This paper reveals for the first time of using degradable microrobots for precise delivery of therapeutic cells in vascular tissue and demonstrates its therapeutic effect in preclinical test.