Folic acid-mesoporous silicon nanoparticles enhance the anticancer activity of the p73-activating small molecule LEM2.

Many drugs with anticancer potential fail in their translation to the clinics due to problems related to pharmacokinetics. LEM2 is a new dual inhibitor of MDM2/mutp53-TAp73 interactions with interesting in vitro anticancer activity, which opens new hopes as an unconventional anticancer therapeutic strategy against cancers lacking p53 or with impaired p53 pathways. As others xanthone derivatives, LEM2 has limited aqueous solubility, posing problems to pursue in vivo assays, and therefore limiting its potential clinical translation. In this work, a mesoporous silicon (PSi)-based nanodelivery system was developed with folate functionalization (APTES-TCPSi-PEG-FA) for targeted delivery, which successfully increased LEM2 solubility when compared to bulk LEM2, evidenced in payload release study. Such effect was reflected on the increase of LEM2 cytotoxicity in HCT116 and MDA-MB-231 cancer cells when treated with LEM2-loaded APTES-TCPSi-PEG-FA, by reducing cell viability lower than 50% in comparison with bulk LEM2. Despite the reduced LEM2 loading degree, which still limits its application in further in vivo assays, the results obtained herein recognize PSi-based nanodelivery systems as a promising strategy to improve LEM2 anticancer activity and bioavailability, which will be relevant for the potential use of this potent TAp73 activator in anticancer therapy.

One-step microfluidics production of enzyme-loaded liposomes for the treatment of inflammatory diseases.

The biopharmaceuticals market is constantly growing. Despite their advantages over the conventional drugs, biopharmaceuticals have short biological half-lifes, which can be increased using liposomes. However, the common bulk methods to produce biopharmaceuticals-loaded liposomes result in lost of encapsulation efficiency (E.E.), resulting in an expensive process. Herein, the encapsulation of a therapeutic enzyme in liposomes is proposed, using a glass-capillary microfluidic technique. Cu,Zn- Superoxide dismutase (SOD) is successfully encapsulated into liposomes (SOD@Liposomes). SOD@Liposomes with a mean size of 135 ± 41 nm, a polydispersity index of 0.13 ± 0.01, an E.E. of 59 ± 6 % and an enzyme activity of 82 ± 3 % are obtained. in vivo experiments show, through an ear edema model, that SOD@Liposomes administered by the intravenous route enable an edema inhibition of 65 % ± 8 %, over the 20 % ± 13 % of SOD in its free form. The histopathological analyses show a higher inflammatory cell accumulation on the ear treated with SOD in its free form, than treated with SOD@Liposomes. Overall, this work highlights the potential of microfluidics for the production of enzyme-loaded liposomes with high encapsulation efficiency, with the intrinsic advantages of the low time-consuming and easily upscaling microfluidic assembly method.

Preparation of cetyl palmitate-based PEGylated solid lipid nanoparticles by microfluidic technique.

In recent years, several studies have shown that the use of solid lipid nanoparticles (SLN) as a colloidal drug delivery system was more advantageous than lipid emulsions, liposomes and polymeric nanoparticles. SLNs have numerous advantages of different nanosystems and rule out many of their drawbacks. Despite the numerous advantages of SLNs, translation from the preclinical formulation to the industrial scale-up is limited. In order to provide a reproducible and reliable method of producing nanoparticles, and thus, obtain an industrial scale-up, several methods of synthesis of nanoparticles by microfluidic have been developed. Microfluidic technique allows a good control and a continuous online synthesis of nanosystems compared to synthesis in bulk, leading to a narrow size distribution, high batch-to-batch reproducibility, as well as to the industrial scale-up feasibility. This work described the optimization process to produce SLNs by microfluidics. The SLNs produced by microfluidics were characterized by complementary optical and morphological techniques and compared with those produced by bulk method. SLNs were loaded with paclitaxel and sorafenib, used as model drugs. The anti-cancer efficiency of the SLNs formulation was estimated with 2D and 3D tumour models of two different cell lines, and the cellular uptake was also studied with fluorescence-assisted measurements.

All-in-one microfluidic assembly of insulin-loaded pH-responsive nano-in-microparticles for oral insulin delivery.

Here, a continuous two-step glass-capillary microfluidic technique to produce a multistage oral delivery system is reported. Insulin is successfully encapsulated into liposomes, which are coated with chitosan to improve their mucoadhesion. The encapsulation in an enteric polymer offers protection from the harsh gastric conditions. Insulin permeability is enhanced across an intestinal monolayer.

Preparation and in vivo evaluation of red blood cell membrane coated porous silicon nanoparticles implanted with 155Tb.

INTRODUCTION: Porous silicon (PSi) nanoparticles are capable of delivering therapeutic payloads providing targeted delivery and sustained release of the payloads. In this work we describe the development and proof-of-concept in vivo evaluation of thermally hydrocarbonized porous silicon (PSi) nanoparticles that are implanted with radioactive 155Tb atoms and coated with red blood cell (RBC) membrane (155Tb-THCPSi). The developed nanocomposites can be utilized as an intravenous delivery platform for theranostic radionuclides. METHODS: THCPSi thin films were implanted with 155Dy ions that decay to 155Tb at the ISOLDE radioactive ion-beam (RIB) facility at CERN. The films were processed to nanoparticles by ball-milling and sonication, and subsequently coated with either a solid lipid and RBC membrane or solely with RBC membrane. The nanocomposites were evaluated in vitro for stability and in vivo for circulation half-life and ex vivo for biodistribution in Balb/c mice. RESULTS: Nanoporous THCPSi films were successfully implanted with 155Tb and processed to coated nanoparticles. The in vitro stability of the particles in plasma and buffer solutions was not significantly different between the particle types, and therefore the RBC membrane coated particles with less laborious processing method were chosen for the biological evaluation. The RBC membrane coating enhanced significantly the blood half-life compared to bare THCPSi particles. In the ex vivo biodistribution study a pronounced accumulation to the spleen was found, with lower uptake in the liver and a minor uptake in the lung, gall bladder and bone marrow. CONCLUSIONS: We have demonstrated, using 155Tb RIB-implanted PSi nanoparticles coated with mouse RBC membranes, the feasibility of using such a theranostic nanosystem for the delivery of RIB based radionuclides with prolonged circulation time. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: For the first time, the RIB implantation technique has been utilized to produce PSi nanoparticle with a surface modified for better persistence in circulation. When optimized, these particles could be used in targeted radionuclide therapy with a combination of chemotherapeutic payload within the PSi structure.

Tumor necrosis factor primes and metal particles activate the NLRP3 inflammasome in human primary macrophages.

Aseptic loosening of total joint replacements is driven by a macrophage-mediated inflammatory reaction to implant-derived wear particles. Phagocytosis of implant debris has been suggested to activate the NLRP3 inflammasome leading to secretion of interleukin (IL)-1ß. However, factors and molecular mechanisms driving the particle-induced inflammasome activation are yet to be fully elucidated. In this study, we investigated the inflammasome response of human primary macrophages to titanium, chromium, and molybdenum particles in vitro. We observed that particles alone were not sufficient to induce IL-1ß secretion, but an additional priming signal-such as bacterial lipopolysaccharide (LPS)-was required to license the inflammasome activation. By using specific inhibitors against the inflammasome signaling pathway, we demonstrate that the particle-induced IL-1ß secretion depended upon activation of the NLRP3 inflammasome. We further hypothesized that tumor necrosis factor (TNF) could substitute for LPS as a priming signal, and found that particle stimulation together with preceding TNF treatment resulted in inflammasome-dependent IL-1ß production as well. Our results show that the NLRP3 inflammasome mediates wear particle responses in human primary macrophages, and its activation does not necessarily require the presence of bacterial components, but can be induced under aseptic conditions by TNF priming. STATEMENT OF SIGNIFICANCE: This study was conducted to elucidate the molecular mechanisms of metal particle-induced IL-1ß secretion in human primary macrophages. Production of this pro-inflammatory mediator from wear particle-activated macrophages has been associated with increased bone loss around total joint replacements-a condition eventually requiring revision surgery. Our results confirm that together with a co-stimulatory priming signal, particles of common implant metals elicit macrophage-mediated IL-1ß secretion through activation of the NLRP3 inflammasome pathway. We also present a concept of TNF priming in this context, demonstrating that the particle-related IL-1ß secretion can take place in a truly sterile environment. Thus, inhibition of inflammasome signaling appears a means to prevent wear particle-induced inflammation and development of peri­prosthetic osteolysis.

Hybrid red blood cell membrane coated porous silicon nanoparticles functionalized with cancer antigen induce depletion of T cells.

Erythrocyte-based drug delivery systems have been investigated for their biocompatibility, long circulation time, and capability to transport cargo all around the body, thus presenting enormous potential in medical applications. In this study, we investigated hybrid nanoparticles consisting of nano-sized autologous or allogeneic red blood cell (RBC) membranes encapsulating porous silicon nanoparticles (PSi NPs). These NPs were functionalized with a model cancer antigen TRP2, which was either expressed on the surface of the RBCs by a cell membrane-mimicking block copolymer polydimethylsiloxane-b-poly-2-methyl-2-oxazoline, or attached on the PSi NPs, thus hidden within the encapsulation. When in the presence of peripheral blood immune cells, these NPs resulted in apoptotic cell death of T cells, where the NPs having TRP2 within the encapsulation led to a stronger T cell deletion. The deletion of the T cells did not change the relative proportion of CD4+ and cytotoxic CD8+ T cells. Overall, this work shows the combination of nano-sized RBCs, PSi, and antigenic peptides may have use in the treatment of autoimmune diseases.

In vitro evaluation of biodegradable lignin-based nanoparticles for drug delivery and enhanced antiproliferation effect in cancer cells.

Currently, nanosystems have been developed and applied as promising vehicles for different biomedical applications. We have developed three lignin nanoparticles (LNPs): pure lignin nanoparticles (pLNPs), iron(III)-complexed lignin nanoparticles (Fe-LNPs), and Fe3O4-infused lignin nanoparticles (Fe3O4-LNPs) with round shape, narrow size distribution, reduced polydispersity and good stability at pH 7.4. The LNPs showed low cytotoxicity in all the tested cell lines and hemolytic rates below 12% after 12 h of incubation. Additionally, they induced hydrogen peroxide production in a small extent and time-dependent manner, and the interaction with the cells increased over time, exhibiting a dose-dependent cell uptake. Concerning the drug loading, pLNPs showed the capacity to efficiently load poorly water-soluble drugs and other cytotoxic agents, e.g. sorafenib and benzazulene (BZL), and improve their release profiles at pH 5.5 and 7.4 in a sustained manner. Furthermore, the BZL-pLNPs presented an enhanced antiproliferation effect in different cells compared to the pure BZL and showed a maximal inhibitory concentration ranging from 0.64 to 12.4 µM after 24 h incubation. Overall, LNPs are promising candidates for drug delivery applications, and the superparamagnetic behavior of Fe3O4-LNPs makes them promising for cancer therapy and diagnosis, such as magnetic targeting and magnetic resonance imaging.

Thermoresponsive Nanoparticles of Self-Assembled Block Copolymers as Potential Carriers for Drug Delivery and Diagnostics.

Thermally responsive hydrogel nanoparticles composed of self-assembled polystyrene-b-poly(N-isopropylacrylamide)-b-polystyrene block copolymers and fluorescent probe 1-anilinonaphthalene-8-sulfonic acid have been prepared by aerosol flow reactor method. We aimed exploring the relationship of intraparticle morphologies, that were, PS spheres and gyroids embedded in PNIPAm matrix, as well PS-PNIPAm lamellar structure, to probe release in aqueous solution below and above the cloud point temperature (CPT) of PNIPAm. The release was detected by fluorescence emission given by the probe binding to bovine serum albumin. Also, the colloidal behavior of hydrogel nanoparticles at varying temperatures were examined by scattering method. The probe release was faster below than above the CPT from all the morphologies of which gyroidal morphology showed the highest release. Colloidal behavior varied from single to moderately aggregated particles in order spheres-gyroids-lamellar. Hydrogel nanoparticles with tunable intra particle self-assembled morphologies can be utilized designing carrier systems for drug delivery and diagnostics.

Polypeptide-based aerosol nanoparticles: self-assembly and control of conformation by solvent and thermal annealing.

Nanoconfined self-assemblies within aerosol nanoparticles and control of the secondary structures are shown here upon ionically complexing poly(L-lysine) (PLL) with dodecylbenzenesulfonic acid (DBSA) surfactant and using solvents chloroform, 1-propanol, or dimethylformamide. Different solvent volatilities and drying temperatures allowed tuning the kinetics of morphology formation. The supramolecular self-assembly and morphology were studied using cryo-TEM and SEM, and the secondary structures, using FT-IR. Highly volatile chloroform led to the major fraction of α-helical conformation of PLL(DBSA), whereas less volatile solvents or higher drying temperatures led to the increasing fraction of ß-sheets. Added drugs budesonide and ketoprofen prevented ß-sheet formation and studied PLL(DBSA)-drug nanoparticles were in the α-helical conformation. Preliminary studies showed that ketoprofen released with a slower rate than budesonide which was hypothesized to result from different localization of drugs within the PLL(DBSA) nanoparticles. These results instruct to prepare polypeptide aerosol nanoparticles with internal self-assembled structures and to control the secondary structures by aerosol solvent annealing, which we foresee to be useful, e.g., toward controlling the release of poorly soluble drug molecules.

Gas-phase synthesis of solid state DNA nanoparticles stabilized by l-leucine.

Aerosol flow reactor is used to generate solid-state nanoparticles in a one-step process that is based on drying of aerosol droplets in continuous flow. We investigated the applicability of aerosol flow reactor method to prepare solid state DNA nanoparticles. Precursor solutions of plasmid DNA with or without complexing agent (polyethylenimine), coating material (l-leucine) and mannitol (bulking material) were dispersed to nanosized droplets and instantly dried in laminar heat flow. Particle morphology, integrity and stability were studied by scanning electron microscopy. The stability of DNA was studied by gel electrophoresis. Plasmid DNA as such degraded in the aerosol flow process. Complexing agent protected DNA from degradation and coating material enabled production of dispersed, non-aggregated, nanoparticles. The resulting nanoparticles were spherical and their mean diameter ranged from 65 to 125nm. The nanoparticles were structurally stable at room temperature and their DNA content was about 10%. We present herein the proof of principle for the production of dispersed solid state nanoparticles with relevant size and intact plasmid DNA.

Coated particle assemblies for the concomitant pulmonary administration of budesonide and salbutamol sulphate.

The aims were to prepare stable and well-dispersible pulmonary fine powders composed of combination drugs with different water solubility, to facilitate concomitant release of corticosteroid budesonide and short acting ß-agonist salbutamol sulphate and to improve the dissolution of the budesonide. The budesonide nanosuspensions were prepared by a wet milling which were mixed then with salbutamol sulphate, mannitol (bulking material) and leucine (coating material) for the preparation of micron-sized particles by an aerosol flow reactor wherein leucine formed a rough coating layer on particle surface. The stable and intact particle assemblies showed excellent aerosolization performance. The emitted doses from the inhaler, Easyhaler(®), were ~3 mg/dose with a coefficient variation of 0.1, and the fine particle fractions were ~50%. Complete dissolution of budesonide nanocrystals from the particles took place within 20 min with the same rate as salbutamol sulphate. Combining the two formulation technologies enabled the encapsulation of drugs with different solubility into a single, intact particle. The leucine coating provided excellent aerosolization properties which allowed fine powder delivery from the inhaler without carrier particles. This study showed the feasibility of preparing powders for combination therapy that are utilized, for instance, in inhalation therapy.

Thermally sensitive block copolymer particles prepared via aerosol flow reactor method: Morphological characterization and behavior in water.

This work describes properties of thermo-sensitive submicron sized particles having the same chemical composition but different morphologies. These particles have been prepared with an aerosol technique using dimethylformamide solutions of linear polystyrene-block-poly(N-isopropylacrylamide-block-polystyrene, PS-b-PNIPAM-b-PS. The particles were characterized by cryo-electron microscopy, microcalorimetry, and light scattering. Block-copolymers self-assembled within the particles forming onion-like, gyroid-like, and spherical morphologies having poly(N-isopropylacrylamide) matrix and physically cross-linking polystyrene domains. The particles were dispersed in aqueous media and their behavior in water was studied both below and above the lower critical solution temperature of poly(N-isopropylacrylamide). We found out that the particles with spherical and gyroid-like morphologies swell considerably in water at 20 °C, whereas at 40 °C the particles resemble more of those studied without water treatment. Light scattering experiments showed that the particles gradually aggregate and precipitate with time at 40 °C. Microcalorimetric studies revealed for all three studied morphologies that PNIPAM undergoes a two-step transition due to the different hydration levels of PNIPAM inside and outside the particles. Thicknesses of the PS and PNIPAM layers within the onion-like particles were analyzed using the TEM micrographs by fitting a model of electron density to the integrated electron intensity data. The surface layer of the particles was found out to be PNIPAM, which was supported by light scattering and microcalorimetry. It was also found out from the TEM micrograph analysis that the width of the outmost PS layer is considerably thinner than the one in the dry state prior to immersion in water, and a degradation scheme is proposed to explain these results.

Intact nanoparticulate indomethacin in fast-dissolving carrier particles by combined wet milling and aerosol flow reactor methods.

PURPOSE: Drug development is often hindered by a drug's low dissolution rate. We present a method to increase dissolution rate of a drug powder by producing crystalline nanoparticles that are dispersed in carrier microparticles. METHODS: Indomethacin crystals of a few hundred nanometers are prepared by media milling using poloxamer 188 as a stabilizer. Nanoparticles are embedded into microparticles with a mannitol matrix and an L-leucine coating layer using an aerosol flow reactor method. RESULTS: Microparticles stabilize the primary nanoparticles in an intact crystalline form and release them when re-dispersed in aqueous medium. Secondary microparticle structure dissolves rapidly, resulting in a fast release and dissolution of indomethacin. In this manner, it is possible to change the surface layer of the particles from the one needed for nanoparticle production to one more suitable for process formulation of pharmaceuticals for, e.g., tablet or pulmonary products. CONCLUSIONS: Particle assemblies where nano-sized crystalline drug domains are embedded in solid microparticles are presented. The present work is a promising approach towards a "nanos-in-micros" concept as a tool for pharmaceutical nanoparticle processing.