Although nanomedicine has been highly investigated for cancer treatment over the past decades, only a few nanomedicines are currently approved and in the market; making this field poorly represented ...in clinical applications. Key research gaps that require optimization to successfully translate the use of nanomedicines have been identified, but not addressed; among these, the lack of control of the release pattern of therapeutics is the most important. To solve these issues with currently used nanomedicines (e.g., burst release, systemic release), different strategies for the design and manufacturing of nanomedicines allowing for better control over the therapeutic release, are currently being investigated. The inclusion of stimuli-responsive properties and prolonged drug release have been identified as effective approaches to include in nanomedicine, and are discussed in this paper. Recently, smart sustained release nanoparticles have been successfully designed to safely and efficiently deliver therapeutics with different kinetic profiles, making them promising for many drug delivery applications and in specific for cancer treatment. In this review, the state-of-the-art of smart sustained release nanoparticles is discussed, focusing on the design strategies and performances of polymeric nanotechnologies. A complete list of nanomedicines currently tested in clinical trials and approved nanomedicines for cancer treatment is presented, critically discussing advantages and limitations with respect to the newly developed nanotechnologies and manufacturing methods. By the presented discussion and the highlight of nanomedicine design criteria and current limitations, this review paper could be of high interest to identify key features for the design of release-controlled nanomedicine for cancer treatment.
CD44 is a potentially rewarding target in cancer therapy, although its mechanisms of ligand binding and internalization are still poorly understood. In this study, we have established quantitative ...relationships between CD44 expression in differently polarized macrophages (M0, M1, and M2‐polarized THP‐1 human macrophages) and the uptake of hyaluronic acid (HA)‐based materials, which are potentially usable for CD44 targeting. We have validated a robust method for macrophage polarization, which sequentially uses differentiating and polarizing factors, and allows to show that CD44 expression depends on polarization (M1 > M0 ≥ M2). It is noteworthy that THP‐1 M2 expressed CD44v6, suggesting their suitability as a model of tumor‐associated macrophages. In the uptake of HA, both as a soluble polymer and in the form of (siRNA‐loaded) nanoparticles, CD44 expression correlated positively with binding, but negatively with internalization. Counterintuitively, it appears that a higher presence of CD44 (in M1) allows a more efficient capture of HA materials, but a lower expression (in M2) is conducive to better internalization. Although possibly cell‐specific, this unexpected relationship indicates that the common paradigm “higher CD44 expression = better targetability” is too simplistic; mechanistic details of both receptor presentation and association still need to be elucidated for a predictable targeting behavior.
This study addresses how the expression of CD44 affects the binding and internalization of hyaluronic acid‐based materials, by using the polarization of human macrophages to vary the expression of this receptor. The results suggest that higher CD44 expression (classical activation) does correlate with enhanced binding, but lower expression levels (non‐polarized and alternative activation) promote better internalization.
This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are ...composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP-HA exchange. The templated chitosan/HA nanoparticles therefore have a mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio (
/
) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.
Coumarins possess a wide array of therapeutic capabilities, but often with unclear mechanism of action. We tested a small library of 18 coumarin derivatives against human invasive breast ductal ...carcinoma cells with the capacity of each compound to inhibit cell proliferation scored, and the most potent coumarin analogues selected for further studies. Interestingly, the presence of two prenyloxy groups (5,7-diprenyloxy-4-methyl-coumarin,
4g
) or the presence of octyloxy substituent (coumarin
4d
) was found to increase the potency of compounds in breast cancer cells, but not against healthy human fibroblasts. The activity of potent compounds on breast cancer cells cultured more similarly to the conditions of the tumour microenvironment was also investigated, and increased toxicity was observed. Results suggest that tested coumarin derivatives could potentially reduce the growth of tumour mass. Moreover, their use as (combination) therapy in cancer treatment might have the potential of causing limited side effects.
Graphic abstract
The mechanical properties of the cellular microenvironment play a crucial role in modulating cell function, and many pathophysiological processes are accompanied by variations in extracellular matrix ...(ECM) stiffness. Lysyl oxidase (LOx) is one of the enzymes involved in several ECM-stiffening processes. Here, we engineered poly(ethylene glycol) (PEG)-based hydrogels with controlled mechanical properties in the range typical of soft tissues. These hydrogels were functionalized featuring free primary amines, which allows an additional chemical LOx-responsive behavior with increase in crosslinks and hydrogel elastic modulus, mimicking biological ECM-stiffening mechanisms. Hydrogels with elastic moduli in the range of 0.5-4 kPa were obtained after a first photopolymerization step. The increase in elastic modulus of the functionalized and enzyme-responsive hydrogels was also characterized after the second-step enzymatic reaction, recording an increase in hydrogel stiffness up to 0.5 kPa after incubation with LOx. Finally, hydrogel precursors containing HepG2 (bioinks) were used to form three-dimensional (3D)
models to mimic hepatic tissue and test PEG-based hydrogel biocompatibility. Hepatic functional markers were measured up to 7 days of culture, suggesting further use of such 3D models to study cell mechanobiology and response to dynamic variation of hydrogels stiffness. The results show that the functionalized hydrogels presented in this work match the mechanical properties of soft tissues, allow dynamic variations of hydrogel stiffness, and can be used to mimic changes in the microenvironment properties of soft tissues typical of inflammation and pathological changes at early stages (e.g., fibrosis, cancer).
The adaptation of inkjet printing technology for the realisation of controlled micro- and nano-scaled biological structures is of great potential in tissue and biomaterial engineering. In this paper ...we present the Olivetti BioJet system and its applications in tissue engineering and cell printing. BioJet, which employs a thermal inkjet cartridge, was used to print biomolecules and living cells. It is well known that high stresses and forces are developed during the inkjet printing process. When printing living particles (i.e., cell suspensions) the mechanical loading profile can dramatically damage the processed cells. Therefore computational models were developed to predict the velocity profile and the mechanical load acting on a droplet during the printing process. The model was used to investigate the role of the stiffness of the deposition substrate during droplet impact and compared with experimental investigations on cell viability after printing on different materials. The computational model and the experimental results confirm that impact forces are highly dependent on the deposition substrate and that soft and viscous surfaces can reduce the forces acting on the droplet, preventing cell damage. These results have high relevance for cell bioprinting; substrates should be designed to have a good compromise between substrate stiffness to conserve spatial patterning without droplet coalescence but soft enough to absorb the kinetic energy of droplets in order to maintain cell viability.
Piezoelectric ceramics, such as BaTiO3, have gained considerable attention in bone tissue engineering applications thanks to their biocompatibility, ability to sustain a charged surface as well as ...improve bone cells' adhesion and proliferation. However, the poor processability and brittleness of these materials hinder the fabrication of three-dimensional scaffolds for load bearing tissue engineering applications. For the first time, this study focused on the fabrication and characterisation of BaTiO3 composite scaffolds by using a multi-material 3D printing technology. Polycaprolactone (PCL) was selected and used as dispersion phase for its low melting point, easy processability and wide adoption in bone tissue engineering. The proposed single-step extrusion-based strategy enabled a faster and solvent-free process, where raw materials in powder forms were mechanically mixed and subsequently fed into the 3D printing system for further processing.
PCL, PCL/hydroxyapatite and PCL/BaTiO3 composite scaffolds were successfully produced with high level of consistency and an inner architecture made of seamlessly integrated layers. The inclusion of BaTiO3 ceramic particles (10% wt.) significantly improved the mechanical performance of the scaffolds (54 ± 0.5 MPa) compared to PCL/hydroxyapatite scaffolds (40.4 ± 0.1 MPa); moreover, the presence of BaTiO3 increased the dielectric permittivity over the entire frequency spectrum and tested temperatures. Human osteoblasts Saos-2 were seeded on scaffolds and cellular adhesion, proliferation, differentiation and deposition of bone-like extracellular matrix were evaluated. All tested scaffolds (PCL, PCL/hydroxyapatite and PCL/BaTiO3) supported cell growth and viability, preserving the characteristic cellular osteoblastic phenotype morphology, with PCL/BaTiO3 composite scaffolds exhibiting higher mineralisation (ALP activity) and deposited bone-like extracellular matrix (osteocalcin and collagen I).
The single-step multi-material additive manufacturing technology used for the fabrication of electroactive PCL/BaTiO3 composite scaffolds holds great promise for sustainability (reduced material waste and manufacturing costs) and it importantly suggests PCL/BaTiO3 scaffolds as promising candidates for load bearing bone tissue engineering applications to solve unmet clinical needs.
•BaTiO3 composites scaffolds were successfully fabricated via a single-step extrusion 3D printing system.•3D scaffolds exhibited a seamless structure with high degree of fidelity to the CAD design.•The incorporation of BaTiO3 improved the mechanical and electroactive properties of polymer-based scaffolds.•BaTiO3 scaffolds promoted cells adhesion, proliferation, and a distinctive deposition of osteocalcin and collagen I.
Rapid prototyping techniques are widely used to fabricate well-defined three-dimensional structures of tissue homologs. The piston-assisted microsyringe (PAM2) is a rapid prototyping technology ...specifically developed for low-shear stress extrusion of viscous hydrogel solutions containing cells. In this article the working parameters of the system were established to guarantee the realization of spatially controlled hydrogel scaffolds. Moreover the shear stresses acting on the cell membrane during extrusion was investigated through a computational fluid-dynamic analysis. The computational models show that the shear stress on the cells is of the order of 100 Pa during the extrusion process. HepG2 cells encapsulated in alginate were then extruded into spatially organized hepatic lobule-like architectures and their viability and function were evaluated. The results show that the metabolic fingerprint of the cells is preserved with respect to controls and the cells are uniformly distributed through the gel scaffold.
A great deal of effort is being dedicated to the development of new devices able to conduct effective in vitro toxicology analyses. This paper describes the use of a microfluidic gradient maker for ...the toxicological analysis of two conventional local anesthetics, bupivacaine and lidocaine on cell cultures. The microfluidic device was designed and simulated using COMSOL Multiphysics
® and the concentration gradient in the microfluidic network was analysed through a fluidodynamic and diffusive study.
Subsequently the device was fabricated with soft lithography, casting PDMS in a master to obtain channels about 250
μm deep.
Both drugs were tested on C2C12 myoblasts and an analysis was performed using propidium iodide staining followed by an imaging processing routine to obtain quantitative dose–response profiles in the gradient maker. The system was critically compared with microwell-based toxicity testing. The results show that the GM is a more sensitive method for detection of cell toxicity, and compared with testing of drug toxicity using microwells with individual cell cultures, allows one shot testing with a single cell culture exposed to a large number of concentrations. However, the flow rates required to obtain a suitable concentration range across the device may damage shear sensitive cells.
The invasion of a matrix by migrating cells is a key step in its remodelling. At least in 2D migration models, cells tend to localize in stiffer areas (durotaxis). Here, we show that mechanical ...properties affect differently the 3D migration rate: non-proteolytic 3D cell migration is facilitated in softer matrices. In these gels, the modulus was varied by introducing defects in fibres, leaving largely intact the nanostructure. The matrices derive from fibrin via functionalization with a bioinert polymer poly(ethylene glycol), PEG through an affinity mechanism identical to that presiding to fibrin own self-assembly. Peptidic end groups on PEG were used to bind fibrinogen globular D regions GPRP (glycine-proline-arginine-proline) for a holes, GHRP (glycine-histidine-arginine-proline) for b holes; Kd evaluated via isothermal titration calorimetry or fluorescence anisotropy. In a dose-dependent manner, both PEGylated peptides decreased gel stiffness, but most other properties at a macroscopic e.g., overall elastic character, strain hardening, and high (>0.5) Poisson ratio or nano/micro level (fibre dimension and pore size) were largely unaffected, suggesting that the softening effect was due to the introduction of defects within fibres, rather than to differences in the network architecture. In these matrices, the key determinant of fibroblast migration was found to be the elastic modulus, rather than the identity or the dose of the PEGylated peptide; softer materials allowed a faster invasion, even if this meant a higher content of non-adhesive PEG. This does not conflict with fibroblast durotaxis (where stiffness controls accumulation but not necessarily the speed of migration) and indicates a way to fine tune the speed of cell colonization.
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