Three-dimensional Multicellular Tumor Spheroids Research Articles

Aptamer Conjugate-Based Ratiometric Fluorescent Probe for Precise Imaging of Therapy-Induced Cancer Senescence.

Therapy-induced cellular senescence has been increasingly recognized as a key mechanism to promote various aspects of carcinogenesis in a nonautonomous manner. Thus, real-time imaging monitoring of cellular senescence during cancer therapy is imperative not only to further elucidate its roles in cancer progression but also to provide guidance for medical management of cancer. However, it has long been a challenging task due to the lack of effective imaging molecule tools with high specificity and accuracy toward cancer senescence. Herein, we report the rational design, synthesis, and evaluation of an aptamer conjugate-based ratiometric fluorescent probe for precise imaging of therapy-induced cancer senescence. Unlike traditional senescence imaging systems, our probe targets two senescence-associated markers at both cellular and subcellular dimensions, namely, aptamer-mediated membrane marker recognition for active cell targeting and lysosomal marker-triggered ratiometric fluorescence changes of two cyanine dyes for site-specific, high-contrast imaging. Moreover, such a two-channel fluorescence response is activated after a one-step reaction and at the same location, avoiding the diffusion-caused signal decay previously encountered in dual-marker activated probes, contributing to spatiotemporally specific imaging of therapy-induced cancer senescence in living cells and three-dimensional multicellular tumor spheroids. This work may offer a valuable tool for a basic understanding of cellular senescence in cancer biology and interventions.

Study of the mechanism of ultrasound-induced enhanced therapeutic effects of a chitosan-based nanoplatform

Ultrasound (US) has been used in drug delivery systems for controlling drug release and activation of US-sensitive drugs for sonodynamic therapy of cancer. In our previous work, we found that erlotinib-grafted chitosan nanocomplexes loading perfluorooctyl bromide and hematoporphyrin under US irradiation showed satisfactory therapeutic effects for non-small cell lung cancer treatment. However, the underlying mechanism of US-mediated delivery and therapy has not been fully explored. In this work, the underlying mechanisms of the US-induced effects of the nanocomplexes were evaluated at the physical and biological levels after the chitosan-based nanocomplexes were characterized. The results showed that US could activate the cavitation effects and promote nanocomplexes penetrating into the depth of three-dimensional multicellular tumor spheroids (3D MCTSs) when nanocomplexes were selectively uptaken by targeted cancer cells, but push the extracellular nanocomplexes out of the 3D MCTSs. US demonstrated strong tissue penetration ability to effectively induce obvious reactive oxygen species production deep inside the 3D MCTSs. Under the US condition of 0.1 W cm−2 for 1 min, US caused little mechanical damage and weak thermal effect to avoid severe cell necrosis, whereas cell apoptosis could be induced by collapse of mitochondrial membrane potential and the nucleus damage. The present study indicates that US can potentially be used jointly with nanomedicine to improve targeted drug delivery and combination therapy of deep-seated tumors.

Mitochondria-targeted cyclometalated iridium-β-carboline complexes as potent non-small cell lung cancer therapeutic agents.

Natural products and metals play a crucial role in cancer research and the development of antitumor drugs. We designed and synthesized three new carboline-based cyclometalated iridium complexes [Ir(C-N)2(PPβC)](PF6), where PPβC=N-(1,10-phenanthrolin-5-yl)-1-phenyl-9H-pyrido[3,4-b]indole-3-carboxamide, C-N=2-phenylpyridine (ppy, Ir1), 2-(2,4-difluorophenyl) pyridine (dfppy, Ir2), 7,8-benzoquinoline (bzq, Ir3), by combining iridium with β-carboline derivative. These iridium complexes exhibited high potential antitumor effects after being promptly taken up by A549 cells. Accumulating in mitochondria rapidly and preferentially, Ir1-3 caused a series of changes in mitochondrial events, including the loss of mitochondrial membrane potential, the depletion of cellular ATP, and the elevation of reactive oxygen species, leading to significant death of A549 cells. Moreover, the activation of intracellular caspase pathway and apoptosis was further validated to contribute to iridium complexes-induced cytotoxicity. These novel iridium complexes exerted a prominent inhibitory effect on tumor growth in a three-dimensional multicellular tumor spheroid model.

Liposomal Binuclear Ir(III)-Cu(II) Coordination Compounds with Phosphino-Fluoroquinolone Conjugates for Human Prostate Carcinoma Treatment.

Novel heteronuclear IrIII-CuII coordination compounds ([Ir(η5-Cp*)Cl2Pcfx-Cu(phen)](NO3)·1.75(CH3OH)·0.75(H2O) (1), [Ir(η5-Cp*)Cl2Pnfx-Cu(phen)](NO3)·1.75(CH3OH)·0.75(H2O) (2), [Ir(η5-Cp*)Cl2Plfx-Cu(phen)](NO3)·1.3(H2O)·1.95(CH3OH) (3), [Ir(η5-Cp*)Cl2Psfx-Cu(phen)] (4)) bearing phosphines derived from fluoroquinolones, namely, sparfloxacin (Hsfx), ciprofloxacin (Hcfx), lomefloxacin (Hlfx), and norfloxacin (Hnfx), have been synthesized and studied as possible anticancer chemotherapeutics. All compounds have been characterized by electrospray ionization mass spectrometry (ESI-MS), a number of spectroscopic methods (i.e., IR, fluorescence, and electron paramagnetic resonance (EPR)), cyclic voltammetry, variable-temperature magnetic susceptibility measurements, and X-ray diffractometry. The coordination geometry of IrIII in all complexes adopts a characteristic piano-stool geometry with the η5-coordinated and three additional sites occupied by two chloride and phosphine ligands, while CuII ions in complexes 1 and 2 form a distorted square-pyramidal coordination geometry, and in complex 3, the coordination geometry around CuII ions is a distorted octahedron. Interestingly, the crystal structure of [Ir(η5-Cp*)Cl2Plfx-Cu(phen)] features the one-dimensional (1D) metal-organic polymer. Liposomes loaded with redox-active and fluorescent [Ir(η5-Cp*)Cl2Pcfx-Cu(phen)] (1L) have been prepared to increase water solubility and minimize serious systemic side effects. It has been proven, by confocal microscopy and an inductively coupled plasma mass spectrometry (ICP-MS) analysis, that the liposomal form of compound 1 can be effectively accumulated inside human lung adenocarcinoma and human prostate carcinoma cells with selective localization in nuclei. A cytometric analysis showed dominance of apoptosis over the other cell death types. Furthermore, the investigated nanoformulations induced changes in the cell cycle, leading to S phase arrest in a dose-dependent manner. Importantly, in vitro anticancer action on three-dimensional (3D) multicellular tumor spheroids has been demonstrated.

Design, Synthesis, and Preclinical Activity in Ovarian Cancer Models of New Phosphanegold(I)-N-heterocyclic Carbene Complexes.

A new series of seven gold(I) complexes (1-7) containing 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene (IPr) and phosphane ligands (L1-L7) were synthesized and evaluated for antitumor activity in ovarian cancer (OvCa) models. The synthesized complexes were characterized by IR, mass spectrometry and NMR spectroscopy, and complex 6 was characterized by XRD crystallography. The antiproliferative effect of the new complexes (1-7) was found to be higher than cisplatin and auranofin in OvCa cells sensitive and resistant to cisplatin. The anticancer activity of the most active complex 6 was investigated using OvCa in vitro models, including three-dimensional (3D) multicellular tumor spheroids and in vivo tumor xenografts. Both cisplatin and auranofin were used for comparative purposes. Complex 6 induced apoptosis, mitochondrial reactive oxygen species, and DNA damage; caused a G1 phase cell cycle arrest, inhibited proteasome activity, and cell migration; modified actin polymerization; and significantly inhibited OvCa murine xenografts. These promising results suggest further preclinical testing of these complexes for future applications.

Dual-Stage Irradiation of Size-Switchable Albumin Nanocluster for Cascaded Tumor Enhanced Penetration and Photothermal Therapy.

The triple-negative breast cancer (TNBC) microenvironment makes a feature of aberrant vasculature, high interstitial pressure, and compact extracellular matrix, which combine to reduce the delivery and penetration of therapeutic agents, bringing about incomplete elimination of cancer cells. Herein, employing the tumor penetration strategy of size-shrinkage combined with ligand modification, we constructed a photothermal nanocluster for cascaded deep penetration in tumor parenchyma and efficient eradication of TNBC cells. In our approach, the photothermal agent indocyanine green (ICG) is laded in human serum albumin (HSA), which is cross-linked by a thermally labile azo linker (VA057) and then further modified with a tumor homing/penetrating tLyP-1 peptide (HP), resulting in a TNBC-targeting photothermal-responsive size-switchable albumin nanocluster (ICG@HSA-Azo-HP). Aided by the enhanced permeability and retention effect and guidance of HP, the ca. 149 nm nanoclusters selectively accumulate in the tumor site and then, upon mild irradiation with the 808 nm laser, disintegrate into 11 nm albumin fractions that possess enhanced intratumoral diffusion ability. Meanwhile, HP initiates the CendR pathway among the nutrient-deficient tumor cells and facilitates the transcellular delivery of the nanocluster and its disintegrated fractions for subsequent therapy. By employing this size-shrinkage and peptide-initiated transcytosis strategy, ICG@HSA-Azo-HP possesses excellent penetration capabilities and shows extensive penetration depth in three-dimensional multicellular tumor spheroids after irradiation. Moreover, with a superior photothermal conversion effect, the tumor-penetrating nanocluster can implement effective photothermal therapy throughout the tumor tissue under a second robust irradiation. Both in vivo orthotopic and ectopic TNBC therapy confirmed the efficient tumor inhibition of ICG@HSA-Azo-HP after dual-stage irradiation. The synergistic penetration strategy of on-demanded size-shrinkage and ligand guidance accompanied by clinically feasible NIR irradiation provides a promising approach for deep-penetrating TNBC therapy.

Multicellular Tumor Spheroids in Nanomedicine Research: A Perspective.

Multicellular tumor spheroids are largely exploited in cancer research since they are more predictive than bi-dimensional cell cultures. Nanomedicine would benefit from the integration of this three-dimensional in vitro model in screening protocols. In this brief work, we discuss some of the issues that cancer nanomedicine will need to consider in the switch from bi-dimensional to three-dimensional multicellular tumor spheroid models.

Improving Photodynamic Therapy Anticancer Activity of a Mitochondria-Targeted Coumarin Photosensitizer Using a Polyurethane–Polyurea Hybrid Nanocarrier

Integration of photosensitizers (PSs) within nanoscale delivery systems offers great potential for overcoming some of the “Achiles’ heels” of photodynamic therapy (PDT). Herein, we have encapsulated a mitochondria-targeted coumarin PS into amphoteric polyurethane–polyurea hybrid nanocapsules (NCs) with the aim of developing novel nanoPDT agents. The synthesis of coumarin-loaded NCs involved the nanoemulsification of a suitable prepolymer in the presence of a PS without needing external surfactants, and the resulting small nanoparticles showed improved photostability compared with the free compound. Nanoencapsulation reduced dark cytotoxicity of the coumarin PS and significantly improved in vitro photoactivity with red light toward cancer cells, which resulted in higher phototherapeutic indexes compared to free PS. Importantly, this nanoformulation impaired tumoral growth of clinically relevant three-dimensional multicellular tumor spheroids. Mitochondrial photodamage along with reactive oxygen species (ROS) photogeneration was found to trigger autophagy and apoptotic cell death of cancer cells.

A mitochondria-localized oxygen self-sufficient two-photon nano-photosensitizer for ferroptosis-boosted photodynamic therapy under hypoxia

Photodynamic therapy (PDT) is a medical technique that has received increasing attention over the last years for the treatment of cancer due to its high spatiotemporal selectivity and non-invasive nature. However, its therapeutic potential is strongly limited due to the hypoxic environment found in solid tumors which is hampering the production of therapeutically active species. To overcome this drawback, herein, the functionalization of graphitic carbon nitride nanosheets with mitochondria-targeting iridium(III) polypyridine complexes for oxygen self-sufficient two-photon PDT is reported. The nanomaterial was found to synergistically generate O2 from endogenous H2O2 and H2O as well as to produce a mixture of 1O2, •OH, and •O2- therapeutic species, presenting the ability to tackle hypoxic tumor microenvironments and intervene by a multimodal mechanism through combined type I and type II two-photon PDT. Capitalizing on this, the nanosheets demonstrated to cause significant cellular damage under hypoxic conditions towards monolayer cancer cells as well as three-dimensional multicellular tumor spheroids by combined apoptosis and ferroptosis. The nanomaterial showed to eradicate a hypoxic human melanoma cancer tumor inside a mouse model upon two-photon irradiation at 750 nm within a single treatment. To the best of our knowledge, this study presents the first example of iridium complex functionalized graphitic carbon nitride nanosheets as photosensitizers for the treatment of hypoxic tumors upon clinically relevant two-photon irradiation.

Multicellular tumor spheroids bridge the gap between two-dimensional cancer cells and solid tumors: The role of lipid metabolism and distribution

Previous studies demonstrated that three-dimensional (3D) multicellular tumor spheroids (MCTS) could more closely mimic solid tumors than two-dimensional (2D) cancer cells in terms of the spatial structure, extracellular matrix-cell interaction, and gene expression pattern. However, no study has been reported on the differences in lipid metabolism and distribution among 2D cancer cells, MCTS, and solid tumors. Here, we used HepG2 liver cancer cell lines to establish these three cancer models. The variations of lipid profiles and spatial distribution among them were explored by using mass spectrometry-based lipidomics and matrix-assisted laser desorption/ionization mass spectrometry imaging (MSI). The results revealed that MCTS, relative to 2D cells, had more shared lipid species with solid tumors. Furthermore, MCTS contained more comparable characteristics than 2D cells to solid tumors with respect to the relative abundance of most lipid classes and mass spectra patterns. MSI data showed that 46 of 71 lipids had similar spatial distribution between solid tumors and MCTS, while lipids in 2D cells had no specific spatial distribution. Interestingly, most of detected lipid species in sphingolipids and glycerolipids preferred locating in the necrotic region to the proliferative region of solid tumors and MCTS. Taken together, our study provides the evidence of lipid metabolism and distribution demonstrating that MCTS are a more suitable in vitro model to mimic solid tumors, which may offer insights into tumor metabolism and microenvironment.

A nanosensitizer self-assembled from oleanolic acid and chlorin e6 for synergistic chemo/sono-photodynamic cancer therapy

BackgroundSono-photodynamic therapy (SPDT) which is the combination of photodynamic therapy (PDT) and sonodynamic therapy (SDT), could exert much better anti-cancer effects than monotherapy. The combination of chemotherapy and PDT or SDT has shown great potential for cancer treatment. However, the combination of SPDT and chemotherapy for cancer treatment is rarely explored. PurposeWe utilized a natural hydrophobic anti-cancer drug oleanolic acid (OA) and a photosensitizer chlorin e6 (Ce6) through self-assembly technology to form a carrier-free nanosensitizer OC for combined chemotherapy and SPDT for cancer treatment. No studies involving using carrier-free nanomedicine for combined chemotherapy/SPDT have been reported yet. Study designAfter fully characterization of OC, the in vitro and in vivo anti-cancer activities of OC were investigated and the mechanisms of the synergistic therapeutic effects were studied. MethodsOC were synthesized through self-assembly technology and characterized by dynamic light scattering (DLS) and an atomic force microscope (AFM). Confocal microscope was used to investigate the intracellular uptake efficiency and the penetration ability of OC. The cell viability of PC9 and 4T1 cells treated with OC under laser and ultrasound (US) irradiation was determined by MTT assay. Furthermore, flow cytometry was performed to detect the reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (MMP), cell apoptosis and cell cycle arrest. Finally, the anti-tumor therapeutic efficacy of OC was investigated in orthotopic 4T1 breast tumor-bearing mouse model. ResultsOC showed an average particle size of around 100 nm with excellent light stability. OC increased more than 23 times accumulation of Ce6 in cancer cells and had strong tumor penetration ability in three-dimensional (3D) multicellular tumor spheroids (MCTSs). Compared with other therapeutic options, OC showed obvious synergistic inhibitory effects under light and US irradiation in PC9 and 4T1 cells with a significant decrease in IC50 values. Mechanism studies showed that OC could generate high ROS, induce MMP loss, and cause apoptosis and cell cycle arrest. In vivo studies also approved the synergistic therapeutic effects of OC in 4T1 mouse models. ConclusionSelf-assembled carrier-free nanosensitizer OC could be a promising therapeutic agent for synergistic chemo/sono-photodynamic therapy for cancer treatment.

An intelligent hypoxia-relieving chitosan-based nanoplatform for enhanced targeted chemo-sonodynamic combination therapy on lung cancer

The clinical efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs)-based targeted molecular therapies (TMT) is inevitably hampered by the development of acquired drug resistance in non-small cell lung cancer (NSCLC) treatment. Sonodymanic therapy (SDT) is a promising new cancer treatment approach, but its effects are restricted by tumor hypoxia. Herein, a nanoplatform fabricated by erlotinib-modified chitosan loading sonosensitizer hematoporphyrin (HP) and oxygen-storing agent perfluorooctyl bromide (PFOB), namely CEPH, was developed to deliver HP to erlotinib-sensitive cells. CEPH with ultrasound could alleviate hypoxia inside the three-dimensional multicellular tumor spheroids, suppress NSCLC cell growth under normoxic or hypoxic condition, and enhance TMT/SDT synergistic effects through elevated production of reactive oxygen species, decrease of mitochondrial membrane potential, and down-regulation of the expression of the proteins EGFR, p-EGFR, and HIF-1α. Hence, CEPH could be a potential nanoplatform to improve the efficacy of oxygen-dependent SDT and overcome hypoxia-induced TMT resistance for enhanced synergistic TMT/SDT.

Mass spectrometry imaging revealed alterations of lipid metabolites in multicellular tumor spheroids in response to hydroxychloroquine

Three-dimensional (3D) multicellular tumor spheroids (MCTS) that mimic the complex tumor microenvironment provide a good platform for in vitro study of drug and endogenous metabolites. Hydroxychloroquine (HCQ) has shown anti-tumor activity in a variety of tumor models. However, the effect of the drug on the alteration of lipid metabolism spatial composition and distribution in the MCTS model is not clear. Herein, we utilized matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in the analysis of A549 lung cancer multicellular spheroids to investigate the in situ spatial distribution of HCQ and its effect on lipid metabolism. We have successfully observed the spatial variations of HCQ in the inner region of the spheroid at different drug-treated time points. The MSI results also demonstrated that HCQ treatment altered the spatial composition of lipids in the inner and outer regions of treated spheroids. Furthermore, the lipidomic results showed that the identified phosphatidylcholines (PC), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), lysophosphatidylethanolamines (LPE), phosphatidylinositols (PI), ceramides (Cer), glucosylceramides (CerG), and diglycerides (DG) were significantly up-regulated, and phosphatidylglycerol (PG) and triglycerides (TG) were remarkable down-regulated. MSI method combined with LC-MS/MS profiling of endogenous metabolites can obtain more detailed information about how spheroids respond to drug and spatial distribution information, thus fostering a better understanding of the relationship between drug-altered lipid metabolism and cancer microenvironment.

AXTEX-4D: A Three-Dimensional Ex Vivo Platform for Preclinical Investigations of Immunotherapy Agents.

The latest advancements in oncology are majorly focused on immuno-oncology (I-O) therapies. However, only ∼7% of drugs are being approved from the preclinical discovery phase to phase 1. The most challenging issues in I-O are the development of active and efficient drugs in an economically feasible way and in a comparatively short time for testing and validation. This mandates an urgent need for the upgradation of preclinical screening models that closely mimic the in vivo tumor microenvironment (TME). The established and most common methods for investigating the tumoricidal activity of I-O drugs are either two-dimensional systems or primary tumor cells in standard tissue culture vessels. Unfortunately, they do not mimic the TME. Consequently, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell-mediated responses in reaction to the administration of I-O drugs. Despite many advantages of multicellular spheroids, challenges (e.g., incompatibility of quantitative assays with spheroid platforms) are still involved in the tedious procedures required for the spheroid culture that is holding back the biological community from adapting the well-recognized spheroid tissue models for studying drug delivery more widely. To this end, we have demonstrated the utility of the 3D ex vivo oncology model, developed on our novel AXTEX-4D™ platform to assess therapeutic efficacies of I-O drugs by investigating immune cell proliferation, migration, infiltration, cytokine profiling, and cytotoxicity of tumor tissueoids. The platform eliminates the need for additional biomolecules such as hydrogels and instead relies on the cancer cells themselves to create their own gradients and microenvironmental factors. In effect, the more comprehensive and ex vivo-like immune-oncology model developed on AXTEX-4D platform can be utilized for high-throughput screening of immunotherapeutic drugs.

Enhanced radiotherapy efficacy of breast cancer multi cellular tumor spheroids through in-situ fabricated chitosan-zinc oxide bio-nanocomposites as radio-sensitizing agents

Overwhelming evidence has shown that three-dimensional multicellular tumor spheroids (MCTSs) as a mimic of in-vivo tumor can accurately exhibit cellular responses to treatments. So, we compared the capability of pure zinc oxide nanoparticles (ZnO-NPs) and chitosan-ZnO bio-nanocomposites (CS-ZnO BNCs) for enhancing the radiosensitization of MDA-MB-231 breast cancer cells (BCCs) in the 3D-MCTSs model. ZnO-NPs and CS-ZnO BNCs were synthesized by a facile co-precipitation method. FE-SEM images revealed that the uniform spherical ZnO-NPs with an average diameter of 35 nm were successfully dispersed on chitosan. MDA-MB-231 MCTSs which were formed in a non-adherent culture plate, possessed functional features of in-vivo tumor. The priority of such culture method to conventionally used 2D monolayer (or parental) cell culture is the mimicking of tumor microenvironment. The toxicity of CS-ZnO BNCs and ZnO-NPs against the MDA-M−231 BCCs was evaluated using MTT-colorimetric assay, which demonstrated superior biocompatibility of CS-ZnO BNCs compared to pure ZnO-NPs (even at high concentration of 100 μg/mL). Survival fraction analysis of cells under clinical X-ray irradiation (6 MV) showed that MCTSs had a higher radioresistance compared to parental cells. Besides, the clonogenic potential of irradiated MCTSs was significantly decreased by the addition of CS-ZnO BNCs similar to that of monolayer cells. The sensitivity enhancement ratios (SER) for MCTSs and monolayer cells were calculated 1.5 and 1.63, respectively. Further, tracking of radiobiological properties and apoptosis induction of MCTSs showed that CS-ZnO BNCs not only could lead to the creation of higher radiation-induced complex DNA break and apoptosis death in MCTSs, but also weakened DNA repair mechanisms. It was found that non-toxic concentration of CS-ZnO BNCs has promising potential to enhance radiosensitivity of resistant-MCTSs as a superior in-vitro tumor model. So, CS-ZnO BNCs can be a prominent candidate for overcoming the resistance of BCCs to radiotherapy.

Enhanced Penetrability of a Tetrahedral Framework Nucleic Acid by Modification with iRGD for DOX-Targeted Delivery to Triple-Negative Breast Cancer.

Poor penetrability and nonselective distribution of chemotherapeutic drugs are the main obstacles for chemotherapy for triple-negative breast cancer (TNBC). In our work, we developed a DNA-based drug delivery system to surmount these barriers. In addition, a tetrahedral framework nucleic acid (tFNA) was employed to load doxorubicin (DOX) with iRGD decoration to form a novel nanoparticle (tFNA/DOX@iRGD). The RGD sequence and the CendR motif in iRGD are used in tumor targeting and tissue penetration, respectively. Based on the sustained serum stability and pH-sensitive release behavior of DOX, tFNA/DOX@iRGD exhibited superiority for biomedical application. Moreover, tFNA/DOX@iRGD showed excellent deep penetration and drug accumulation in three-dimensional (3D) multicellular tumor spheroids compared to DOX and tFNA/DOX. Additionally, the therapeutic effect was verified in a 4T1 subcutaneous tumor model, and the complexes displayed a superior antitumor and antiangiogenic efficiency with fewer collateral damages. Therefore, these findings suggested that tFNA/DOX@iRGD might be a more effective pattern for drug delivery and TNBC therapy.

Os(II)-Bridged Polyarginine Conjugates: The Additive Effects of Peptides in Promoting or Preventing Permeation in Cells and Multicellular Tumor Spheroids.

The preparation of two polyarginine conjugates of the complex Os(II) [bis-(4′-(4-carboxyphenyl)-2,2′:6′,2″-terpyridine)] [Os-(Rn)2]x+ (n = 4 and 8; x = 10 and 18) is reported, to explore whether the R8 peptide sequence that promotes cell uptake requires a contiguous amino acid sequence for membrane permeation or if this can be accomplished in a linearly bridged structure with the additive effect of shorter peptide sequences. The conjugates exhibit NIR emission centered at 754 nm and essentially oxygen-insensitive emission with a lifetime of 89 ns in phosphate-buffered saline. The uptake, distribution, and cytotoxicity of the parent complex and peptide derivatives were compared in 2D cell monolayers and a three-dimensional (3D) multicellular tumor spheroid (MCTS) model. Whereas, the bis-octaarginine sequences were impermeable to cells and spheroids, and the bis-tetraarginine conjugate showed excellent cellular uptake and accumulation in two 2D monolayer cell lines and remarkable in-depth penetration of 3D MCTSs of pancreatic cancer cells. Overall, the data indicates that cell permeability can be promoted via non-contiguous sequences of arginine residues bridged across the metal centre.

Optimizing the Polymer Cloak for Upconverting Nanoparticles: An Evaluation of Bioactivity and Optical Performance.

The ability of upconversion nanoparticles (UCNPs) to convert low-energy near-infrared (NIR) light into high-energy visible-ultraviolet light has resulted in their development as novel contrast agents for biomedical imaging. However, UCNPs often succumb to poor colloidal stability in aqueous media, which can be conquered by decorating the nanoparticle surface with polymers. The polymer cloak, therefore, plays an instrumental role in ensuring good stability in biological media. This study aims to understand the relationship between the length and grafting density of the polymer shell on the physicochemical and biological properties of these core-shell UCNPs. Poly(ethylene glycol) methyl ether methacrylate block ethylene glycol methacrylate phosphate (PPEGMEMAn-b-PEGMP3) with different numbers of PEGMEMA repeating units (26, 38, and 80) was prepared and attached to the UCNPs via the phosphate ligand of the poly(ethylene glycol methacrylate phosphate) (PEGMP) block at different polymer densities. The in vitro and in vivo protein corona, cellular uptake in two-dimensional (2D) monolayer and three-dimensional (3D) multicellular tumor spheroid (MCTS) models, and in vivo biodistribution in mice were evaluated. Furthermore, the photoluminescence of single-polymer-coated UCNPs was compared in solid state and cancer cells using laser scanning confocal microscopy (LSCM). Our results showed that the bioactivity and luminescence properties are chain length and grafting density dependent. The UCNPs coated with the longest PPEGMEMA chain, grafted at low brush density, were able to reduce the formation of the protein corona in vitro and in vivo, while these UCNPs also showed the brightest upconversion luminescence in the solid state. Moreover, these particular polymer-coated UCNPs showed enhanced cellular uptake, extended in vivo blood circulation time, and more accumulation in the liver, brain, and heart.

Lactobionic acid-modified thymine-chitosan nanoparticles as potential carriers for methotrexate delivery

In order to achieve efficient delivery of methotrexate (MTX), thymine-chitosan nanoparticles (Thy-Cs NPs) were prepared, and further decorated with lactobionic acid (LA) to obtain tumor-targeting nanoparticles (LA-Thy-Cs NPs). These nanoparticles possessed a regular spherical structure with the average size about 190–250 nm and narrow size distribution, which were kinetically stable in the physiological environment. Due to electrostatic interactions and multiple hydrogen-bonding interactions between MTX and carriers, MTX was loaded into Thy-Cs NPs with high drug loading content (~20%). MTX release from Thy-Cs NPs was significantly accelerated in the mildly acidic environment due to the destruction of two types of non-covalent interactions. In vitro cell experiments demonstrated that LA-Thy-Cs NPs could be efficiently internalized into hepatoma carcinoma cells, leading to higher cytotoxicity. Moreover, MTX-loaded LA-Thy-Cs NPs performed an enhanced growth inhibition in three-dimensional multicellular tumor spheroids. Thus, the LA decorated thymine-chitosan nanocarriers can be a promising candidate for efficient delivery of MTX.

Investigating the influence of block copolymer micelle length on cellular uptake and penetration in a multicellular tumor spheroid model.

The efficient penetration of drug nanocarriers into tumors is an important prerequisite for therapeutic and diagnostic success. The physicochemical properties of nanocarriers, including size, shape and surface chemistry have been shown to influence their transport in biological systems. Recent studies have shown that elongated nanoparticles (NPs) can exhibit advantageous properties in comparison to spherical NPs, but these experiments have involved a variety of different materials, many of which are characterized by a broad size distribution. Here we describe a series of rigid rod-like micelles of uniform width, with narrow length distributions, and common surface chemistry, and examine their cell uptake and penetration into multicellular tumor spheroids (MCTSs) formed from two human breast cancer cell lines (MDA-MB-436 and MDB-MB-231). These micelles were prepared from a polyferrocenylsilane (PFS) diblock copolymer (BCP) with a corona block consisting of a statistical polymer of aminopropyl methacrylamide and oligo(ethyleneglycol methacrylate) (PFS27-b-PAPMA3-stat-POEGMA48). The rigid rod micelles, with a common width (12 nm) and lengths ranging from 80 to 2000 nm, were prepared by seeded growth crystallization-driven self-assembly in ethanol and then transferred to water. To consider whether changing the shape of these micelles affects its uptake and penetration behavior, analogous spherical micelles were prepared by direct nanoprecipitation into water. Both micelle shape and length were found to influence cellular uptake and penetration into 500 μm MCTSs. Laser confocal fluorescence microscopy was used to examine penetration of these micelles into three-dimensional MCTS up to 90 μm depth. Micelles with an elongated shape and short length (80 nm) demonstrated the deepest penetration into the MCTSs formed by MDA-MB-436 cells. Micelles with lengths of 200 nm also showed substantial penetration into these MCTS, but the extent and depth of tumor penetration of the rod-like micelles decreased with increasing aspect ratio. MCTS of MDA-MB-231 cells had a less dense, more open structure than those formed by MDA-MB-436 cells. Here more extensive penetration was observed, particularly for the longer micelle samples.