Cancer-Targeting Nanoparticles for Combinatorial Nucleic Acid Delivery

Nucleic acids are a promising type of therapeutic for the treatment of a wide range of conditions, including cancer, but they also pose many delivery challenges. For efficient and safe delivery to cancer cells, nucleic acids must generally be packaged into a vehicle, such as a nanoparticle, that will allow them to be taken up by the target cells and then released in the appropriate cellular compartment to function. As with other types of therapeutics, delivery vehicles for nucleic acids must also be designed to avoid unwanted side effects; thus, the ability of such carriers to target their cargo to cancer cells is crucial. Classes of nucleic acids, hurdles that must be overcome for effective intracellular delivery, types of nonviral nanomaterials used as delivery vehicles, and the different strategies that can be employed to target nucleic acid delivery specifically to tumor cells are discussed. Additonally, nanoparticle designs that facilitate multiplexed delivery of combinations of nucleic acids are reviewed.     

Cargo-towing fuel-free magnetic nanoswimmers for targeted drug delivery

Fuel-free nanomotors are essential for future in-vivo biomedical transport and drug-delivery applications. Herein, the first example of directed delivery of drug-loaded magnetic polymeric particles using magnetically driven flexible nanoswimmers is described. It is demonstrated that flexible magnetic nickel-silver nanoswimmers (5-6 μm in length and 200 nm in diameter) are able to transport micrometer particles at high speeds of more than 10 μm s(-1) (more than 0.2 body lengths per revolution in dimensionless speed). The fundamental mechanism of the cargo-towing ability of these magnetic (fuel-free) nanowire motors is modelled, and the hydrodynamic features of these cargo-loaded motors discussed. The effect of the cargo size on swimming performance is evaluated experimentally and compared to a theoretical model, emphasizing the interplay between hydrodynamic drag forces and boundary actuation. The latter leads to an unusual increase of the propulsion speed at an intermediate particle size. Potential applications of these cargo-towing nanoswimmers are demonstrated by using the directed delivery of drug-loaded microparticles to HeLa cancer cells in biological media. Transport of the drug carriers through a microchannel from the pick-up zone to the release microwell is further illustrated. It is expected that magnetically driven nanoswimmers will provide a new approach for the rapid delivery of target-specific drug carriers to predetermined destinations.     

An Ultra pH‐Sensitive and Aptamer‐Equipped Nanoscale Drug‐Delivery System for Selective Killing of Tumor Cells

Nanotechnology has often been applied in the development of targeted drug‐delivery systems for the treatment of cancer. An ideal nanoscale system for drug delivery should be able to selectively deliver and rapidly release the carried therapeutic drug(s) in cancer cells and, more importantly, not react to off‐target cells so as to eliminate unwanted toxicity on normal tissues. To reach this goal, a selective chemotherapeutic is formulated using a hollow gold nanosphere (HAuNS) equipped with a biomarker‐specific aptamer (Apt), and loaded with the chemotherapy drug doxorubicin (DOX). The formed Apt‐HAuNS‐Dox, approximately 42 nm in diameter, specifically binds to lymphoma tumor cells and does not react to control cells that do not express the biomarker. Through aptamer‐mediated selective cell binding, the Apt‐HAuNS‐Dox is internalized exclusively into the targeted tumor cells, and then released the DOX intracellularly. Of note, although the formed Apt‐HAuNS‐Dox is stable under normal biological conditions (pH 7.4), it appears ultrasensitive to pH change and rapidly releases 80% of the loaded DOX within 2 h at pH 5.0, a condition seen in cell lysosomes. Functional assays using cell mixtures show that the Apt‐HAuNS‐Dox selectively kills lymphoma tumor cells, but has no effect on the growth of the off‐target cells in the same cultures, indicating that this ultra pH‐sensitive Apt‐HAuNS‐Dox can selectively treat cancer through specific aptamer guidance, and will have minimal side effects on normal tissue.     

NIR Photoresponsive Crosslinked Upconverting Nanocarriers Toward Selective Intracellular Drug Release

An NIR‐responsive mesoporous silica coated upconverting nanoparticle (UCNP) conjugate is developed for controllable drug delivery and fluorescence imaging in living cells. In this work, antitumor drug doxorubicin (Dox) molecules are encapsulated within cross‐linked photocaged mesoporous silica coated UCNPs. Upon 980 nm light irradiation, Dox could be selectively released through the photocleavage of theo‐nitrobenzyl (NB) caged linker by the converted UV emission from UCNPs. This NIR light‐responsive nanoparticle conjugate demonstrates high efficiency for the controlled release of the drug in cancer cells. Upon functionalization of the nanocarrier with folic acid (FA), this photocaged FA‐conjugated silica‐UCNP nanocarrier will also allow targeted intracellular drug delivery and selective fluorescence imaging towards the cell lines with high level expression of folate receptor (FR).     

Cytokine Secreting Microparticles Engineer the Fate and the Effector Functions of T‐Cells

T‐cell immunotherapy is a promising approach for cancer, infection, and autoimmune diseases. However, significant challenges hamper its therapeutic potential, including insufficient activation, delivery, and clonal expansion of T‐cells into the tumor environment. To facilitate T‐cell activation and differentiation in vitro, core–shell microparticles are developed for sustained delivery of cytokines. These particles are enriched by heparin to enable a steady release of interleukin‐2 (IL‐2), the major T‐cell growth factor, over 10+ d. The controlled delivery of cytokines is used to steer lineage specification of cultured T‐cells. This approach enables differentiation of T‐cells into central memory and effector memory subsets. It is shown that the sustained release of stromal cell‐derived factor 1α could accelerate T‐cell migration. It is demonstrated that CD4+ T‐cells could be induced to high concentrations of regulatory T‐cells through controlled release of IL‐2 and transforming growth factor beta. It is found that CD8+ T‐cells that received IL‐2 from microparticles are more likely to gain effector functions as compared with traditional administration of IL‐2. Culture of T‐cells within 3D scaffolds that contain IL‐2‐secreting microparticles enhances proliferation as compared with traditional, 2D approaches. This yield a new method to control the fate of T‐cells and ultimately to new strategies for immune therapy.     

A Simply Modified Lymphocyte for Systematic Cancer Therapy

Cytotherapy has received considerable attention in the field of cancer therapy, and various chemical or genetic methods have been applied to remold natural cells for improved therapeutic outcome of cytotherapy. A simple method to modify lymphocytes for cancer treatment by using a clinically used molecule, δ‐aminolevulinic acid (δ‐ALA), is reported here. After incubation with this molecule, tumor‐targeted lymphocytes spontaneously synthesize anti‐neoplastic drug protoporphyrin X (PpIX), and specifically accumulate in cancer tissue. Under periodic 630 nm laser irradiation, lymphocytes generate vesicle‐like apoptotic body (Ab) containing the above‐produced PpIX, and the facilitated delivery of PpIX from Ab makes an excellent therapeutic effect for Ras‐mutated cancer cells under a second irradiation. Importantly, a microfluidic device is further fabricated to simplify cell sorting and drug synthesis with a one‐step operation, which could promote generalization of this strategy. In vitro and in vivo studies confirm the success of such an easy‐operated and global‐regulated strategy for cancer therapy.     

Cancer‐Cell‐Specific Induction of Apoptosis Using Mesoporous Silica Nanoparticles as Drug‐Delivery Vectors

Targeted delivery of the chemotherapeutic agent methotrexate (MTX) to cancer cells using poly(ethyleneimine)‐functionalized mesoporous silica particles as drug‐delivery vectors is reported. Due to its high affinity for folate receptors, the expression of which is elevated in cancer cells, MTX serves as both a targeting ligand and a cytotoxic agent. Enhanced cancer‐cell apoptosis (programmed cell death) relative to free MTX is thus observed at particle concentrations where nonspecific MTX‐induced apoptosis is not observed in the nontargeted healthy cell line, while corresponding amounts of free drug affect both cell lines equally. The particles remain compartmentalized in endo‐/lysosomes during the time of observation (up to 72 h), while the drug is released from the particle only upon cell entry, thereby inducing selective apoptosis in the target cells. As MTX is mainly attached to the particle surface, an additional advantage is that the presented carrier design allows for adsorption (loading) of additional drugs into the pore network for therapies based on a combination of drugs.     

Porous Silicon Nanoneedles Modulate Endocytosis to Deliver Biological Payloads

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane‐nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state‐of‐the‐art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin‐coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae‐ and clathrin‐mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle‐mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell‐manipulation technologies.     

Indium-tin oxide coated microfabricated device for the injection of a single cell into a fused silica capillary for chemical cytometry

A microfabricated device is described for the capture and injection of a single mammalian cell into a fused silica capillary for subsequent analysis by chemical cytometry. The device consists of a 500 μm diameter well made from polydimethylsiloxane on an indium-tin oxide coated microscope slide. The bottom of the well contains a 2 μm high aperture, which was designed to block passage of cells. A cellular suspension was allowed to settle on the device, and aspiration through the aperture was used to trap a single NG-108 cell. Untrapped cells were washed from the device, and a 150 μm outer diameter and 50 μm inner diameter capillary was placed in the well. To inject a cell, voltage was applied to the indium-tin oxide while simultaneously applying vacuum at the distal end of the capillary.     

Fluorinated Polymer Mediated Transmucosal Peptide Delivery for Intravesical Instillation Therapy of Bladder Cancer

Surgical intervention combined with intravesical instillation of chemotherapeutics to clear residual cancer cells after operation is the current standard treatment method for bladder cancer. However, the poor bioavailability of active pharmaceutical ingredients for bladder cancer cells on account of the biological barriers of bladder mucosa, together with significant side effects of currently used intravesical medicine, have limited the clinical outcomes of localized adjuvant therapy for bladder cancer. Aiming at improved intravesical instillation therapy of bladder cancer, a fluorinated polyethylenimine (F‐PEI) is employed here for the transmucosal delivery of an active venom peptide, polybia‐mastoparan I (MPI), which shows selective antiproliferative effect against various bladder cancer cell lines. Upon simple mixing, MPI and F‐PET would coassemble to form stable nanoparticles, which show greatly improved cross‐membrane and transmucosal penetration capacities compared with MPI alone or nonfluorinated MPI/PEI nanoparticles. MPI/F‐PEI shows higher in vivo tumor growth inhibition efficacy for local treatment of a subcutaneous tumor model. More excitingly, as further demonstrated in an orthotopic bladder cancer model, MPI/F‐PEI offers remarkably improved therapeutic effects compared to those achieved by free MPI or the first‐line bladder cancer drug mitomycin C. This work presents a new transmucosal delivery carrier particularly promising for intravesical instillation therapy of bladder cancer.     

Photosensitizer‐Incorporated Quadruplex DNA‐Gated Nanovechicles for Light‐Triggered,Targeted Dual Drug Delivery to Cancer Cells

A novel light‐operated vehicle for targeted intracellular drug delivery is constructed using photosensitizer‐incorporated G‐quadruplex DNA‐capped mesoporous silica nanoparticles. Upon light irradiation, the photosensitizer generates ROS, causing the DNA capping to be cleaved and allowing cargo to be released. Importantly, this platform makes it possible to develop a drug‐carrier system for the synergistic combination of chemotherapy and PDT for cancer treatment with spatial/temporal control. Furthermore, the introducing of targeting ligands further improves tumor targeting efficiency. The excellent biocompatibility, cell‐specific intracellular drug delivery, and cellular uptake properties set up the basis for future biomedical application that require in vivo controlled, targeted drug delivery.     

Combinatorial Drug Conjugation Enables Nanoparticle Dual‐Drug Delivery

A new approach to loading multiple drugs onto the same drug‐delivery nanocarrier in a precisely controllable manner, by covalently preconjugating multiple therapeutic agents through hydrolyzable linkers to form drug conjugates, is reported. In contrast to loading individual types of drugs separately, this drug‐conjugates strategy enables the loading of multiple drugs onto the same carrier with a predefined stoichiometric ratio. The cleavable linkers allow the therapeutic activity of the individual drugs to be resumed after the drug conjugates are delivered into the target cells and unloaded from the delivery vehicle. As a proof of concept, the synthesis and characterization of paclitaxel–gemcitabine conjugates are demonstrated. The time‐dependent hydrolysis kinetics and cytotoxicity of the combinatorial drug conjugates against human pancreatic cancer cells are examined. It is shown that the synthesized drug conjugates can be readily encapsulated into a lipid‐coated polymeric drug‐delivery nanoparticle, which significantly improves the cytotoxicity of the drug conjugates as compared to the free drug conjugates.     

Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging

Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single‐chain anti‐EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR‐expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR‐QDs or ScFvEGFR‐IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular‐targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR‐targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.     

MOFBOTS: Metal–Organic‐Framework‐Based Biomedical Microrobots

Motile metal?organic frameworks (MOFs) are potential candidates to serve as small‐scale robotic platforms for applications in environmental remediation, targeted drug delivery, or nanosurgery. Here, magnetic helical microstructures coated with a kind of zinc‐based MOF, zeolitic imidazole framework‐8 (ZIF‐8), with biocompatibility characteristics and pH‐responsive features, are successfully fabricated. Moreover, it is shown that this highly integrated multifunctional device can swim along predesigned tracks under the control of weak rotational magnetic fields. The proposed systems can achieve single‐cell targeting in a cell culture media and a controlled delivery of cargo payloads inside a complex microfluidic channel network. This new approach toward the fabrication of integrated multifunctional systems will open new avenues in soft microrobotics beyond current applications.     

Targeted Liposome‐Loaded Microbubbles for Cell‐Specific Ultrasound‐Triggered Drug Delivery

One of the main problems in cancer treatment is disease relapse through metastatic colonization, which is caused by circulating tumor cells (CTCs). This work reports on liposome‐loaded microbubbles targeted to N‐cadherin, a cell–cell adhesion molecule expressed by CTCs. It is shown that such microbubbles can indeed bind to N‐cadherin at the surface of HMB2 cells. Interestingly, in a mixture of cells with and without N‐cadherin expression, binding of the liposome‐loaded microbubbles mainly occurs to the N‐cadherin‐expressing cells. Importantly, applying ultrasound results in the intracellular delivery of a model drug (loaded in the liposomes) in the N‐cadherin‐expressing cells only. As described in this paper, such liposome‐loaded microbubbles may find application as theranostics and in devices aimed for the specific killing of CTCs in blood.     

Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (～99%) for MCF-7 cells and very high recovery rate (～89%) for DMS-79 cells, with minimal amounts of leukocytes present.     

Engineering Biomimetic Platesomes for pH‐Responsive Drug Delivery and Enhanced Antitumor Activity

Biomimetic camouflage, i.e., using natural cell membranes for drug delivery, has demonstrated advantages over synthetic materials in both pharmacokinetics and biocompatibility, and so represents a promising solution for the development of safe nanomedicine. However, only limited efforts have been dedicated to engineering such camouflage to endow it with optimized or additional properties, in particular properties critical to a “smart” drug delivery system, such as stimuli‐responsive drug release. A pH‐responsive biomimetic “platesome” for specific drug delivery to tumors and tumor‐triggered drug release is described. This platesome nanovehicle is constructed by merging platelet membranes with functionalized synthetic liposomes and exhibits enhanced tumor affinity, due to its platelet membrane–based camouflage, and selectively releases its cargo in response to the acidic microenvironment of lysosomal compartments. In mouse cancer models, it shows significantly better antitumor efficacy than nanoformulations based on a platesome without pH responsiveness or those based on traditional pH‐sensitive liposomes. A convenient way to incorporate stimuli‐responsive features into biomimetic nanoparticles is described, demonstrating the potential of engineered cell membranes as biomimetic camouflages for a new generation of biocompatible and efficient nanocarriers.     

MSN Anti‐Cancer Nanomedicines: Chemotherapy Enhancement,Overcoming of Drug Resistance,and Metastasis Inhibition

In the anti‐cancer war, there are three main obstacles resulting in high mortality and recurrence rate of cancers: the severe toxic side effect of anti‐cancer drugs to normal tissues due to the lack of tumor‐selectivity, the multi‐drug resistance (MDR) to free chemotherapeutic drugs and the deadly metastases of cancer cells. The development of state‐of‐art nanomedicines based on mesoporous silica nanoparticles (MSNs) is expected to overcome the above three main obstacles. In the view of the fast development of anti‐cancer strategy, this review highlights the most recent advances of MSN anti‐cancer nanomedicines in enhancing chemotherapeutic efficacy, overcoming the MDR and inhibiting metastasis. Furthermore, we give an outlook of the future development of MSNs‐based anti‐cancer nanomedicines, and propose several innovative and forward‐looking anti‐cancer strategies, including tumor tissue?cell?nuclear successionally targeted drug delivery strategy, tumor cell‐selective nuclear‐targeted drug delivery strategy, multi‐targeting and multi‐drug strategy, chemo‐/radio‐/photodynamic‐/ultrasound‐/thermo‐combined multi‐modal therapy by virtue of functionalized hollow/rattle‐structured MSNs.     

Surface‐Mediated Nucleic Acid Delivery by Lipoplexes Prepared in Microwell Arrays

Many delivery methods have been developed to improve the therapeutic efficacy and facilitate the clinical translation of nucleic acid‐based therapeutics. A facile surface‐mediated nucleic acid delivery by lipoplexes is prepared in a microwell array, which combines the advantages of lipoplexes as an efficient carrier system, surface‐mediated delivery, and the control of surface topography. Uniform disc‐like lipoplexes containing nucleic acids are formed in the microwell array with a diameter of ～818 nm and thickness of ～195 nm. The microwell array‐mediated delivery of lipoplexes containing FAM‐oligodeoxynucleotides is ～18.6 and ～10.6 times more efficient than the conventional transfection method in an adherent cell line (A549 non‐small cell lung cancer cells) and a suspension cell line (KG‐1a acute myelogenous leukemia cells), respectively. MicroRNA‐29b is then used as a model nucleic acid to investigate the therapeutic efficacy of lipoplexes delivered by the microwell array. Compared to conventional transfection methods, the effective therapeutic dosage of microRNA‐29b is reduced from the microgram level to the nanogram level by lipoplexes prepared in the microwell array. The microwell array is also a very flexible platform. Both nucleic acid therapeutics and imaging reagents are incorporated in lipoplexes and successfully delivered to A549 cells, demonstrating its potential applications in theranostic medicine.     

Rapid Production of Cell‐Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform

This study establishes a novel microfluidic platform for rapid encapsulation of cells at high densities in photocrosslinkable microspherical hydrogels including poly(ethylene glycol)‐diacrylate, poly(ethylene glycol)‐fibrinogen, and gelatin methacrylate. Cell‐laden hydrogel microspheres are advantageous for many applications from drug screening to regenerative medicine. Employing microfluidic systems is considered the most efficient method for scale‐up production of uniform microspheres. However, existing platforms have been constrained by traditional microfabrication techniques for device fabrication, restricting microsphere diameter to below 200 µm and making iterative design changes time‐consuming and costly. Using a new molding technique, the microfluidic device employs a modified T‐junction design with readily adjustable channel sizes, enabling production of highly uniform microspheres with cell densities (10–60 million cells mL^?1) and a wide range of diameters (300–1100 µm), which are critical for realizing downstream applications, through rapid photocrosslinking (≈1 s per microsphere). Multiple cell types are encapsulated at rates of up to 1 million cells per min, are evenly distributed throughout the microspheres, and maintain high viability and appropriate cellular activities in long‐term culture. This microfluidic encapsulation platform is a valuable and readily adoptable tool for numerous applications, including supporting injectable cell therapy, bioreactor‐based cell expansion and differentiation, and high throughput tissue sphere‐based drug testing assays.