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Tissue clearing and expansion microscopy have significantly advanced neuroscience by enabling the visualization of spatial information about biological components, from tissue-level architecture to nanoscale structures. Currently, their application is limited to tissues without prolonged fixation, which excludes long-term preserved specimens, such as human brains. To address this issue, we present a new multiplex labeling and expansion-applicable method for centimeter-sized archived tissue (MOCAT). We successfully applied MOCAT to formalin-fixed paraffin-embedded (FFPE) mouse, pig, and human brain blocks up to 15 years old. The reliability of MOCAT was demonstrated by the indistinguishable spatial and quantitative information of biomarkers obtained from FFPE intact mouse brains compared to that from freshly prepared SDS-delipidated intact mouse brains. Additionally, MOCAT-processed FFPE samples exhibited superior long-term antigenicity preservation and suitability for advanced expansion microscopy in human brain compared to those kept in 10% formalin. In summary, MOCAT combined with FFPE fulfills the demand for long-term tissue preservation and organ-level multiplex imaging in super-resolution.

Current tissue clearing and labeling approaches require freshly prepared samples to avoid problems related to over-fixation, such as loss of antigenicity or difficulty of tissue clearing. Thus, the predicament of specimens with long-term preservation limits the application of state-of-art technology to archived human brain tissues. Here, we present the development and validation of multiplex labeling of centimeter-sized archived tissue (MOCAT)—a formalin-fixed paraffin-embedding (FFPE)–based sample preservation and delipidation technique for centimeter-level tissue clearing and immunolabeling. We demonstrate that MOCAT sufficiently delipidates whole mouse brain and human brain blocks and is superior to 10% formalin for long-term antigenicity preservation; moreover, the spatial information of biomarkers offered by MOCAT-processed intact mouse brains is identical to that offered by freshly prepared SDS-delipidated intact mouse brains. MOCAT not only satisfies the need for long-term tissue storage before performing volumetric multiplex imaging for research purposes but also has the potential to be used in human clinical examination.

T lymphocyte infiltration with immunotherapy potentially suppresses most devastating brain tumors. However, local immune privilege and tumor heterogeneity usually limit the penetration of immune cells and therapeutic agents into brain tumors, leading to tumor recurrence after treatment. Here, a rabies virus glycoprotein (RVG)-camouflaged gold yarnball (RVG@GY) that can boost the targeting efficiency at a brain tumor

Astaxanthin (AST) is a naturally occurring xanthophyll carotenoid having the potential to be used as an anticancer agent; however, the human body has a low bioavailability of AST due to its poor solubility in the water phase. Therefore, we applied D-α-tocopheryl polyethylene glycol succinate (TPGS) as an emulsifier and natural edible peanut oil to form a steady oil-in-water (O/W) nanoemulsion loaded with AST (denoted as TAP-nanoemulsion). TAP-nanoemulsions were stable without the droplet coalescence against thermal treatments (30-90°C), pH value changes (over a range of 2.0-8.0), and ionic strength adjustments (at NaCl concentrations of 100-500 mM) measured by dynamic light scattering (DLS). AST within TAP-nanoemulsion was released up to 80% in a simulated intestinal enzymatic fluid in vitro, and the overall recovery rate was fairly consistent in the Caco-2 cellular model. In order to further evaluate in vivo melanoma inhibitory experiments, we injected the fluorescent-stained B16F10 cells into female C57BL/6 mouse tail veins and treated TAP-nanoemulsion in an oral gavage. qRT-PCR and Western blot demonstrated that TAP-nanoemulsion triggered effectively the apoptosis pathway, including enhancements of cleaved caspase-3 and caspase-9, ataxia-telangiectasia mutated kinase (ATM), and p21WAF1/CIP1 (p21) and decreases of B-cell lymphoma 2 (Bcl-2); cyclins D, D1, and E; mitogen-activated protein kinase (MEK); extracellular signal-regulated kinases (ERK); nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB); and matrix metallopeptidase-1 and metallopeptidase-9 (MMP-1 and MMP-9) in both gene and protein expressions. In conclusion, this study suggests that TAP-nanoemulsion with the oral treatment has a positive chemotherapy effect in melanoma with lung metastases in vivo. As far as we know, this is the first time to demonstrate that an antioxidant in nanoparticle administration cures lung metastatic melanoma.

A donut-shaped nanostructure that can effectively approach lung metastasis and offer a burst penetrative delivery to overcome tumor heterogeneity has been proposed. The intravenously injected donut serves as a tumor-adhesion agent via the margination effect and hierarchical donut-shaped nanostructure, which drives nanoparticle-induced extracellular leakiness (nanoEL) both in vitro and in vivo. In an animal metastasis model, more than 72% of donuts localize to lung metastatic tumors instead of normal tissues. Upon the application of a high-frequency magnetic field (MF), a burst generation of polydox (pDox) particles from the carbon porous reservoirs of donuts enhances the retention in the deep tumor, facilitating penetrative delivery. Via pH-responsive hydrozone bonds, the penetrative pDox particles are cleaved into doxorubicin (Dox), yielding high intracellular concentrations of activated Dox at the tumor. The donut-shaped nanostructure loaded with pDox improved the survival rates in a mouse model of metastatic lung cancer and the activity of immune response.

Drug delivery depots boosting a local concentration of therapeutic agents have received great attention in clinical applications due to their low occurrence of side effects and high therapeutic efficacy. However, once the payload is exhausted, the local drug concentration will be lower than the therapeutic window. To address this issue, an injectable double-strand deoxyribonucleic acid (DNA)-architected nanoraspberry depot (DNR-depot) was developed that can refill doxorubicin (Dox, an anticancer drug) from the blood and remotely control drug release on demand. The large porous surface on a uniform nanoraspberry (NR) filled covalently with DNA serves as a Dox sponge-like refilling reservoir, and the NR serves as a magnetic electrical absorber. Via the strong affinity between Dox and DNA molecules, the refilling process of Dox can be achieved on DNR-depot both in vitro and in vivo. Upon high-frequency magnetic field (HFMF) treatment, the remotely triggered release of Dox is actuated by the dissociation of Dox and DNA molecules, facilitating an approximately 800% improvement in drug concentration at the tumor site compared to free Dox injection alone. Furthermore, the cycles of refilling and release can be carried out more than 3 times in vivo within 21 days. The combination of refilling and HFMF-programmable Dox release in tumors via DNR-depot can effectively inhibit tumor growth for 30 days.

A high-frequency magnetic field (MF) generates an electric current by charging conductors that enable the induction of various biological processes, including changes in cell fate and programming. In this study, we show that electromagnetized carbon porous nanocookies (NCs) under MF treatment facilitate magnetoelectric conversion for growth factor release and cell stimulation to induce neuron cell differentiation and proliferation in vitro and in vivo. Integrating four-dimensional printing technology, the NCs are exposed on the surface, which enhances the cell adhesion and allows direct manipulation of electromagnetic stimulation of the cells. Remarkably, large amounts of growth factor encapsulated in NC@conduit resulted in excellent permeability and on-demand release, improving the in vivo layers of myelin sheaths and directing the axon orientation at 1 month postimplantation. This study offers proof of principle for MF-guided in vivo neuron regeneration as a potentially viable tissue regeneration approach for neuronal diseases. Researchers in Taiwan have developed a printable biomaterial that has potential for neuron regeneration and treating neural diseases. Four-dimensional printed materials are objects created using 3D printing technology but whose properties can be altered in time, by electrical pulses for example. A team led by San-Yuan Chen from National Chiao Tung University and Shang-Hsiu Hu from National Tsing Hua University, both in Hsinchu, made biomedical four-dimensional printed material by combining stretchable and biocompatible graphene oxide nanosheets with so-called nanocookies: porous carbon with silica embedded into its surface. The pores in the carbon act as a scaffold on which neurons can grow, and an electric current induced in the silica by a high-frequency magnetic field stimulated this growth. The team observed this stimulation in both cell cultures and rat models. 4D Printing Conduits: Electromagnitized carbon porous nanoccookies (NCs) under MF facilitate magneto-electrical conversion for growth factor release and cell simulation. Integrating four-dimensional (4D) printed technology, exposed NCs are able to enhances the cell adhesion and manifest directly electromagnetic stimulation to cells.

Zoledronic acid (ZA), a third-generation nitrogen-heterocycle-containing bisphosphonate, has been frequently used as an anti-resorptive agent to treat cancer-involved hypercalcemia and painful bone metastases. In order to expand the clinical applications of ZA toward the extraskeletal tumor treatment, it is essential to develop the functionalized nanocarriers capable of carrying high ZA payload and achieving intracellular triggered ZA release. In this end, the ZA-encapsulated hybrid polymeric nanoparticles were fabricated in this work by co-association of the amphiphilic diblock copolymer poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG), tocopheryl polyethylene glycol succinate (TPGS) segments and ionic complexes composed of ZA molecules and branched poly(ethylenimine) (PEI) segments. Notably, the ionic pairings of PEI segments with ZA molecules not only assisted encapsulation of ZA into the PLGA-rich core of hybrid nanoparticles but also reduced adhesion of ZA on the surfaces of hydrophobic cores, thus largely increasing ZA loading capacity. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) characterization revealed that the ZA/PEI-loaded nanoparticles had a well-dispersed spherical shape. Moreover, compared to short PEI1.8k (1.8 kDa) segments, the longer PEI10k (10 kDa) segments formed more robust complexes with ZA molecules, thus prominently promoting ZA loading content of hybrid nanoparticles and their colloidal stability. Interestingly, with the suspension pH being reduced from 7.4 to 5.0, the considerable disruption of ZA/PEI ionic complexes owing to the acid-activated protonation of ZA molecules and the developed proton sponge-like effect inside the nanoparticle matrix upon the protonated PEI segments facilitated the rapid release of ZA molecules from drug-loaded hybrid nanoparticles. The results of in vitro cellular uptake and cytotoxicity studies showed that the ZA/PEI-loaded hybrid nanoparticles were internalized by MCF-7 cells upon energy-dependent endocytosis and displayed a superior cytotoxic effect to free ZA. This work demonstrates that the unique ZA/PEI-loaded hybrid polymeric nanoparticles display great promise for anticancer applications.

Dual on‐demand delivery of therapeutic cargos and energy by transporters can latently mitigate side effects and provide the unique aspects required for precision medicine. To achieve this goal, metal‐organic frameworks (MOFs), hybrid materials constructed from metal ions and polydentate organic linkers, have attracted attention for controlled drug release and energy delivery in tumors. With appropriate characteristics such as tunable pore size, high surface area, and tailorable composition, therapeutic agents (drug molecules or responsive agents) can be effectively encapsulated in MOFs. Based on their intrinsic properties, many physically or chemically responsive agents are able to achieve precise on‐demand drug release and energy generation (thermal or dynamic therapy) using MOFs (as energy absorbers). Herein, the results obtained with various stimuli‐responsive MOFs (including materials from the Institute Lavoisier [MIL], zeolitic imidazolate frameworks [ZIFs], MOFs from the University of Oslo [UiO], and other MOFs) used for tumor suppression are summarized. Furthermore, with the appropriate stimulus, catalytic therapy (caused by the Fenton reaction induced by MOFs) can be provided via the utilization of existing high levels of H2O2 in cancer cells, which potentially elicits immune responses. In addition, the issues impeding clinical translation are also discussed, including the need to overcome tumor heterogeneity and to recognize the innate immune system and possible effects. As the references reveal, additional comprehensive strategies and studies are needed to enable broad applications and potent translational developments.

Compact nanohybrids can potentially unite various therapeutic features and reduce side effects for precise cancer therapy. However, the poor accumulation and limited tumor penetration of drugs at the tumor impede the manifestation of nanomedicine. We developed a rabies virus glycoprotein (RVG)-amplified hierarchical targeted hybrid that acts as a stealthy and magnetolytic carrier that transports dual tumor-penetrating agents incorporating two drugs (boron-doped graphene quantum dots (B-GQDs)/doxorubicin and pH-responsive dendrimers (pH-Den)/palbociclib). The developed RVG-decorated hybrids (RVG-hybrids) enhance the accumulation of drugs at tumor by partially bypassing the BBB via spinal cord transportation and pH-induced aggregation of hierarchical targeting. The penetrated delivery of dual pH-Den and B-GQD drugs to deep tumors is actuated by magnetoelectric effect, which are able to generate electrons to achieve electrostatic repulsion and disassemble the hybrids into components of a few nanometers in size. The synergy of magnetoelectric drug penetration and chemotherapy was achieved by delivery of the B-GQDs and pH-Den to orthotopic tumors, which prolonged the host survival time. This RVG-amplified dual hierarchical delivery integrated with controlled and penetrated release from this hybrid improve the distribution of the therapeutic agents at the brain tumor for synergistic therapy, exhibiting potential for clinic use.

Injectable hydrogels in regeneration medicine can potentially mimic hierarchical natural living tissue and fill complexly shaped defects with minimally invasive implantation procedures. To achieve this goal, however, the versatile hydrogels that usually possess the nonporous structure and uncontrollable spatial agent release must overcome the difficulties in low cell‐penetrative rates of tissue regeneration. In this study, an adaptable microporous hydrogel (AMH) composed of microsized building blocks with opposite charges serves as an injectable matrix with interconnected pores and propagates gradient growth factor for spontaneous assembly into a complex shape in real time. By embedding gradient concentrations of growth factors into the building blocks, the propagated gradient of the nerve growth factor, integrated to the cell‐penetrative connected pores constructed by the building blocks in the nerve conduit, effectively promotes cell migration and induces dramatic bridging effects on peripheral nerve defects, achieving axon outgrowth of up to 4.7 mm and twofold axon fiber intensity in 4 days in vivo. Such AMHs with intrinsic properties of tunable mechanical properties, gradient propagation of biocues and effective induction of cell migration are potentially able to overcome the limitations of hydrogel‐mediated tissue regeneration in general and can possibly be used in clinical applications.

Dual-targeted delivery of drugs and energy by nanohybrids can potentially alleviate side effects and improve the unique features required for precision medicine. To realize this aim, however, the hybrids which are often rapidly removed from circulation and the piled up tumors periphery near the blood vessels must address the difficulties in low blood half-lives and tumor penetration. In this study, a sponge-inspired carbon composites-supported red blood cell (RBC) membrane that doubles as a stealth agent and photolytic carrier that transports tumor-penetrative agents (graphene quantum dots and docetaxel (GQD-D)) and heat with irradiation was developed. The RBC-membrane enveloped nanosponge (RBC@NS) integrated to a targeted protein that accumulates in tumor spheroids via high lateral bilayer fluidity exhibits an 8-fold increase in accumulation compared to the NS. Penetrative delivery of GQDs to tumor sites is actuated by near-infrared irradiation through a one-atom-thick structure, facilitating penetration and drug delivery deep into the tumor tissue. The synergy of chemotherapy and photolytic effects was delivered by the theranostic GQDs deep into tumors, which effectively damaged and inhibited the tumor in 21 days when treated with a single irradiation. This targeted RBC@GQD-D/NS with the capabilities of enhanced tumor targeting, NIR-induced drug penetration into tumors, and thermal ablation for photolytic therapy promotes tumor suppression and exhibits potential for other biomedical applications.

The synthesis and characterization of an NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an α-cyclodextrin ring that encircles it and locks the payload “cargo” molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this smart biocompatible carrier system showed obvious uptake and consequent release of the drug in tumor cells, could selectively induce the tumor cell death and enhance the capability of inhibition of tumor growth in vivo. The controlled drug delivery system demonstrated its use as a potential theranostic material.

Sub-50 nm nanoparticles feature long circulation and deep tumor penetration. However, at high volume fractions needed for intravenous injection, safe, highly biocompatible phospholipids cannot form such nanoparticles due to the fluidity of phospholipid shells. Here we overcome this challenge using a nano-surfactant, a sterilized 18-amino-acid biomimetic of the amphipathic helical motif abundant in HDL-apolipoproteins. As it induces a nanoscale phase (glass) transition in the phospholipid monolayer, the peptide stabilizes 5–7 nm phospholipid micelles that do not fuse at high concentrations but aggregate into stable micellesomes exhibiting size-dependent penetration into tumors. In mice bearing human Her-2-positive breast cancer xenografts, high-payload paclitaxel encapsulated in 25 nm (diameter) micellesomes kills more cancer cells than paclitaxel in standard clinical formulation, as evidenced by the enhanced apparent diffusion coefficient of water determined by in vivo MR imaging. Importantly, the bio-inertness of this biomimetic nano-surfactant spares the nanoparticles from being absorbed by liver hepatocytes, making them more generally available for drug delivery.

Stimuli-responsive drug-delivery systems constitute an appealing approach to direct and restrict drug release spatiotemporally at the specific site of interest. However, it is difficult for most systems to affect every cancer cell in a tumor tissue due to the presence of the natural tumor barrier, leading to potential tumor recurrence. Here, core-shell magnetoresponsive virus-mimetic nanocapsules (VNs), which can infect cancer cells sequentially and double as a magnetothermal agent fabricated through anchoring iron oxide nanoparticles in a single-component protein (lactoferrin) shell, are reported. With large payload of hydrophilic/hydrophobic anticancer cargos, doxorubicin and palictaxel, VNs can simultaneously give a rapid drug release and intense heat while applying an external high-frequency magnetic field (HFMF). Furthermore, after being liberated from dead cells by HFMF manipulation, the constructive VNs can sequentially infect neighboring cancer cells and deliver sufficient therapeutic agents to next targeted sites. With high efficiency for sequential cell infections, VNs have successfully eliminated subcutaneous tumor after a combinatorial treatment. These results demonstrate that the VNs could be used for locally targeted, on-demand, magnetoresponsive chemotherapy/hyperthermia, combined with repeated cell infections for tumor therapy and other therapeutic applications.

A magneto-responsive energy/drug carrier that enhances deep tumor penetration with a porous nano-composite is constructed by using a tumor-targeted lactoferrin (Lf) bio-gate as a cap on mesoporous iron oxide nanoparticles (MIONs). With a large payload of a gas-generated molecule, perfluorohexane (PFH), and a hydrophobic anti-cancer drug, paclitaxel (PTX), Lf-MIONs can simultaneously perform bursting gas generation and on-demand drug release upon high-frequency magnetic field (MF) exposure. Biocompatible PFH was chosen and encapsulated in MIONs due to its favorable phase transition temperature (56 °C) and its hydrophobicity. After a short-duration MF treatment induces heat generation, the local pressure increase via the gasifying of the PFH embedded in MION can substantially rupture the three-dimensional tumor spheroids in vitro as well as enhance drug and carrier penetration. As the MF treatment duration increases, Lf-MIONs entering the tumor spheroids provide an intense heat and burst-like drug release, leading to superior drug delivery and deep tumor thermo-chemo-therapy. With their high efficiency for targeting tumors, Lf-MIONs/PTX-PFH suppressed subcutaneous tumors in 16 days after a single MF exposure. This work presents the first study of using MF-induced PFH gasification as a deep tumor-penetrating agent for drug delivery.

A polymeric nanovehicle featuring a histidine-rich surface was developed for tumor-targeted delivery of doxorubicin. By tumor acidity-triggered surface charge neutralization and aggregation of drug-carrying nanoparticles, the drug accumulation within the tumor was significantly enhanced.

Combination therapy for cancer patients is an important standard of care protocol because it can elicit synergistic therapeutic effects and reduce systemic toxicity by simultaneously modulating multiple cell-signaling pathways and overcoming multidrug resistance. Nanocarriers are expected to play a major role in delivering multiple drugs to tumor tissues by overcoming biological barriers. However, especially considering the different physical chemistry of chemotherapeutic drugs, it is highly desirable to develop a codelivery nanocarrier for controlled and targeted delivery of both hydrophobic and hydrophilic drugs. This review reports the recent developments in various combinational drug delivery systems and the simultaneous use of combinational drug delivery systems with functional agents.

A novel photo-responsive protein–graphene–protein (PGP) capsule that doubles as a photothermal agent with core/shell structure is constructed by anchoring reduced graphene oxide nanosheets on one-component protein (lactoferrin) shell through a double emulsion method. PGP capsules can transport fully concealed hydrophilic anticancer cargo, doxorubicin (Dox), with a large payload (9.43 μmol g-1) to be later unloaded in a burst-like manner by photo-actuation triggered by near-infrared irradiation. Being biocompatible yet with a high cancer cell targeting efficiency, PGP capsules have successfully eradicated subcutaneous tumors in 10 d following a single 5 min NIR irradiation without distal damage. Besides, the photochemothermal therapy of PGP capsules eradicates tumor cells not only in the light-treating area but also widely light-omitted tumor cells, overcoming the tumor recurrence due to efficient cell killing efficacy. These results demonstrate that the PGP capsule is a potential new drug delivery platform for local-targeting, on-demand, photoresponsive, combined chemotherapy/hyperthermia for tumor treatment and other biomedical applications.

The biocompatible thiol-functionalized rGO SH /PMA SH microcapsules encapsulating nerve growth factor (NGF) are arrayed onto a transparent and conductive substrate, i.e., indium tin oxide (ITO), to integrate electrically stimulated cellular differentiation, electrically controlled NGF release, and topographically rough nano-surfaces into a 3-D platform for nerve regeneration. The rGO SH /PMA SH microcapsules with microscale topography function not only as an adhesive coating to promote the adhesion of PC12 cells but also as electroactive NGF-releasing electrodes that stimulate NGF release and accelerate the differentiation of PC12 cells during electrical stimulation. Once electrical treatment is applied, NGF release and electrically enhanced cellular differentiation lead to an obvious increase both in the percentage of cells with neurites and in the neurite length. This length can reach nearly 90 µm within 2 days of cell culture. The average neurite length is signifi cantly increased (four-fold) after culture on the rGO SH / PMA SH microcapsule substrate for 2 days compared with culture on a substrate without an rGO SH /PMA SH coating. These multifunctional rGO SH / PMA SH microcapsules may be used as potential 3-D patterned substrates for neural regeneration and neural prosthetics in tissue engineering applications.

Nanobubbles can serve as promising, next-generation theranostic platforms for ultrasound (US) and magnetic resonance (MR) imaging, and combined magnetic targeting (MT) and high-intensity focused ultrasound (HIFU)-triggered drug release for tumor therapy. Nanobubble-based dual contrast enhancement agents encapsulated with perfluoropentane and stabilized with superparamagnetic iron oxide (SPIO) nanoparticles have been synthesized through a single-step emulsion process from thermosensitive F127 and polyacrylic acid (PAA). Both US and MR imaging contrast can be optimized by varying the shell thickness and SPIO-embedded concentration. The US contrast can be enhanced from a mean gray value of 62 to 115, and the MR r2 value can be enhanced from 164 to 208 (s(-1)mM (-1)Fe) by increasing the SPIO concentration from 14.1 to 28.2mg/mL, respectively. In vivo investigations of SPIO-embedded nanobubbles in excised tumors under external MT revealed that the US and MR signals change quantitatively compared to the same site without MT. This combined strategy enables the nanobubbles to enhance both passive targeting (increasing the permeability by HIFU) and physical MT of chemotherapeutic drugs to tumors. The integration of functionalities makes this nanobubble system a powerful and viable new tool to achieve simultaneous in vivo tumor imaging and efficacious cancer therapy.

A novel photo-responsive drug carrier that doubles as a photothermal agent with a nanocookie-like structure is constructed by coating amorphous carbon on a mesoporous silica support self-assembled on a sheet of reduced graphene oxide. With a large payload (0.88 mmolg �1 ) of a hydrophobic anticancer drug, (S)-(+)-camptothecin (CPT), nanocookies simultaneously provide a burst-like drug release and intense heat upon near-infrared exposure. Being biocompatible yet with a high effi ciency for cell uptake, nanocookies have successfully eradicated subcutaneous tumors in 14 days following a single 5 min NIR irradiation without distal damage. These results demonstrate that the nanocookie is an excellent new delivery platform for local, on-demand, NIR-responsive, combined chemotherapy/hyperthermia for tumor treatment and other biomedical applications.

Gene therapy holds promise to suppress carcinomas but still remains far removed from clinic because of the lack of a safe and effective delivery system. Besides enhancing transfection efficiency, the difficulty in gene therapy is how to deliver sufficient gene molecules to the site of interest. Herein, the rational design of surfactant-free lipo-polymersomes (LPPs) to overcome these problems is reported, simultaneously using a lipid-stabilized double emulsion approach. The LPPs are designed to conceal the cationic lipids and plasmid DNA inside the core along with iron oxide nanoparticles/polymer interlayer and a relatively neutral lipid shell to avoid the undesired interaction during circulation, leading to high accumulation in the tumors of mice. Furthermore, guided by an external magnetic field and the folic acid (FA) that target tumors via folate receptor-mediated endocytosis on the cell surface, the vectors demonstrate a 30-40-fold increase in cell uptake. Moreover, this synergistic tumor-targeted approach can enhance a 10-fold increase in in vivo transfection efficacy by promoting the delivery of LPPs to cancer cells and facilitating the endosomal escape of gene molecules. The new insights and capabilities represent a major step in nanovector engineering for safe and efficient gene delivery.

Delivery of drug and energy within responsive carriers that effectively target and accumulate in cancer cells promises to mitigate side effects and to enhance the uniquely therapeutic efficacy demanded for personalized medicine. To achieve this goal, however, these carriers, which are usually piled up at the periphery of tumors near the blood vessel, must simultaneously overcome the challenges associated with low tumor penetration and the transport of sufficient cargos to deep tumors to eradicate whole cancer cells. Here, we report a sponge-like carbon material on graphene nanosheet (graphene nanosponge)-supported lipid bilayers (lipo-GNS) that doubles as a photothermal agent and a high cargo payload platform and releases a burst of drug/energy (docetaxel (DTX) and gasified perfluorohexane (PFH)) and intense heat upon near-infrared irradiation. Ultrasmall lipo-GNS (40 nm) modified with a tumor-targeting protein that penetrates tumor spheroids through transcytosis exhibited a 200-fold increase in accumulation relative to a 270 nm variant of the lipo-GNS. Furthermore, a combination of therapeutic agents (DTX and PFH) delivered by lipo-GNS into tumors was gasified and released into tumor spheroids and successfully ruptured and suppressed xenograft tumors in 16 days without distal harm when subjected to a single 10 min near-infrared laser treatment. Moreover, no tumor recurrence was observed over 60 days post-treatment. This sophisticated lipo-GNS is an excellent delivery platform for penetrated, photoresponsive, and combined gasification/chemo-thermotherapy to facilitate tumor treatment and for use in other biological applications.

In this study, double-emulsion capsules (DECs) capable of concealing drug-incorporated targeted-supermolecules are developed to achieve "on-demand" supermolecule release and enhanced sequential targeting for magneto-chemotherapy. These water-in-oil-in-water DECs less than 200 nm in diameter are synthesized using a single component of PVA (polyvinyl alcohol) polymer and the magnetic nanoparticles, which are capable of encapsulating large quantities of targeted supermolecules composed of palitaxel-incorporated beta-cyclodextrin decorated by hyaluronic acid (HA, a CD44-targeting ligand) in the watery core. The release profiles (slow, sustained and burst release) of the targeted supermolecules can be directly controlled by regulating the high-frequency magnetic field (HFMF) and polymer conformation without sacrificing the targeting ability. Through an intravenous injection, the positive targeting of the supermolecules exhibited a 20-fold increase in tumor accumulation via the passive targeting and delivery of DECs followed by positive targeting of the supermolecules. Moreover, this dual-targeting drug-incorporated supermolecular delivery vehicle at the tumor site combined with magneto-thermal therapy suppressed the cancer growth more efficiently than treatment with either drug or supermolecule alone.

Quantum-dot-tagged reduced graphene oxide (QD-rGO) nanocomposites (left) internalized into targeted tumor cells display bright fluorescence from the QDs (right); by absorbing NIR radiation incident on the rGO and converting it into heat, they also cause simultaneous cell death and fluorescence reduction (bottom). The nanocomposite is thus capable of tumor imaging, photothermal therapy and in situ monitoring of treatment in progress.

Graphene and its derivatives have unique physical and chemical properties that make them promising vehicles for photothermal therapy (PTT)-based cancer treatment. With intrinsic near-infrared (NIR) absorption properties, graphene-based nanomaterials can be used for PTT and other therapeutics, particularly in combination therapy, to provide successful thermal ablation of cancer cells. In the recent years, advances in graphene-based PTT have produced efficient and efficacious tumor inhibition via nanomaterial structural design and different functionalizations of graphene-derived nanocomposites. Graphene-based nanosystems exhibit multifunctional properties that are useful for PTT applications including enhancement of multimodalities, guided imaging, enhanced chemotherapy and low-power efficient PTT for optimum therapeutic efficiency. Therefore, in this review, we address critical issues and future aspects of PTT-based combination therapy.