Abdulaziz Almalik

Hyaluronic acid (HA) presentation as a tool to modulate and control the receptor-mediated uptake of HA-coated nanoparticles

The natural turnover of free hyaluronic acid (HA) is predominantly based on its CD44-mediated internalisation in leukocytes. In a phagocytic cell model (RAW 264.7 murine macrophages) we here provide conclusive evidence that this receptor-mediated mechanism endocytosis is responsible also of the uptake of materials where HA is used as a coating agent, in this case chitosan/triphosphate nanoparticles on whose surface HA is electrostatically adsorbed. Alginate-coated nanoparticles were used as a control and they appeared to undergo a qualitatively similar endocytic process, which was mediated by a different scavenging receptor yet to be identified. In this general picture, an important, modulating role appears to be played by how receptors can cluster around individual nanoparticles. The CD44 slow representation (24-48 h) enforces a limit in the amount of available HA internalisation receptors; therefore a higher affinity, and hence a higher degree of clustering, would yield a lower number of internalised nanoparticles. HA presentation can be varied by acting on nanoparticle structure/morphology, and our data suggest that a better presentation may be linked to both higher affinity and lower capacity/uptake rate. Paradoxically, this result would suggest that particles with a lower affinity for CD44 may allow a more efficient HA-mediated delivery of payloads.

Protein-gold clusters-capped mesoporous silica nanoparticles for high drug loading, autonomous gemcitabine/doxorubicin co-delivery, and in-vivo tumor imaging.

Functional nanocarriers capable of transporting high drug contents without premature leakage and to controllably deliver several drugs are needed for better cancer treatments. To address this clinical need, gold cluster bovine serum albumin (AuNC@BSA) nanogates were engineered on mesoporous silica nanoparticles (MSN) for high drug loadings and co-delivery of two different anticancer drugs. The first drug, gemcitabine (GEM, 40wt%), was loaded in positively-charged ammonium-functionalized MSN (MSN-NH3(+)). The second drug, doxorubicin (DOX, 32wt%), was bound with negatively-charged AuNC@BSA electrostatically-attached onto MSN-NH3(+), affording highly loaded pH-responsive MSN-AuNC@BSA nanocarriers. The co-delivery of DOX and GEM was achieved for the first time via an inorganic nanocarrier, possessing a zero-premature leakage behavior as well as drug loading capacities seven times higher than polymersome NPs. Besides, unlike the majority of strategies used to cap the pores of MSN, AuNC@BSA nanogates are biotools and were applied for targeted red nuclear staining and in-vivo tumor imaging. The straightforward non-covalent combination of MSN and gold-protein cluster bioconjugates thus leads to a simple, yet multifunctional nanotheranostic for the next generation of cancer treatments.

Hyaluronic acid-coated chitosan nanoparticles: molecular weight-dependent effects on morphology and hyaluronic acid presentation.

Chitosan nanoparticles are popular carriers for the delivery of macromolecular payloads, e.g. nucleic acids. In this study, nanoparticles were prepared via complexation with triphosphate (TPP) anions and were successively coated with hyaluronic acid (HA). Key variables of the preparative process (e.g. chitosan and HA molecular weight) were optimised in view of the maximisation of loading with DNA, of the Zeta potential and of the dimensional stability, and the resulting particles showed excellent storage stability. We have focused on the influence of chitosan molecular weight on nanoparticle properties. Larger molecular weight increased their porosity (=decreased cross-link density), and this caused also larger dimensional changes in response to variations in osmotic pressure or upon drying. The dependency of nanoparticle porosity on chitosan molecular weight had a profound effect on the adsorption of HA on the nanoparticles; HA was apparently able to penetrate deeply into the more porous high molecular weight (684 kDa) chitosan nanoparticles, while it formed a corona around those composed of more densely cross-linked low molecular weight (25 kDa) chitosan. Atomic Force Microscopy (AFM) allowed not only to highlight the presence of this corona, but also to estimate its apparent thickness to about 20-30 nm (in a dry state). The different morphology has a significant effect on the way HA is presented to biomolecules, and this has specific relevance in relation to interactions with HA receptors (e.g. CD44) that influence kinetics and mechanism of nanoparticle uptake. Finally, it is worth to mention that chitosan molecular weight did not appear to greatly affect the efficiency of nanoparticle loading with DNA, but significantly influenced its chitosanase-triggered release, with high molecular chitosan nanoparticles seemingly more prone to degradation by this enzyme.

HA-Coated Chitosan Nanoparticles for CD44-Mediated Nucleic Acid Delivery

CD44, the main receptor of hyaluronic acid (HA), is overexpressed in several pathological conditions and therefore can be seen as an interesting target for therapeutic intervention. Here, an approach using HA-coated chitosan (CS)-triphosphate (TPP) nanoparticles is investigated, using CS with different molecular weight (25 and 684 kDa), which influences HA presentation, and enzymatic and pH stability. In a study of nuclease stability, post-digestion of nanoparticles with chitosanase reveals that pDNA is at least partially degraded by DNAse; this may suggest that literature results overestimate the polyplex stability against nucleases. Using cells with a significantly different CD44 expression (RAW 264.7 macrophages-high levels; K562 leukemia cells-low levels; Kelly neuroblastoma cells-absent), the selectivity of CD44-mediated transfection is proven. Further, using luciferase pDNA and then later anti-luc siRNA, low MW CS-based nanoparticles show the best results despite a lower internalization efficiency; this effect is ascribed to a more efficient endosomal disruption and nucleic acid de-complexation.

Mesoporous Silica and Organosilica Nanoparticles: Physical Chemistry, Biosafety, Delivery Strategies, and Biomedical Applications

Predetermining the physico-chemical properties, biosafety, and stimuli-responsiveness of nanomaterials in biological environments is essential for safe and effective biomedical applications. At the forefront of biomedical research, mesoporous silica nanoparticles and mesoporous organosilica nanoparticles are increasingly investigated to predict their biological outcome by materials design. In this review, it is first chronicled that how the nanomaterial design of pure silica, partially hybridized organosilica, and fully hybridized organosilica (periodic mesoporous organosilicas) governs not only the physico-chemical properties but also the biosafety of the nanoparticles. The impact of the hybridization on the biocompatibility, protein corona, biodistribution, biodegradability, and clearance of the silica-based particles is described. Then, the influence of the surface engineering, the framework hybridization, as well as the morphology of the particles, on the ability to load and controllably deliver drugs under internal biological stimuli (e.g., pH, redox, enzymes) and external noninvasive stimuli (e.g., light, magnetic, ultrasound) are presented. To conclude, trends in the biomedical applications of silica and organosilica nanovectors are delineated, such as unconventional bioimaging techniques, large cargo delivery, combination therapy, gaseous molecule delivery, antimicrobial protection, and Alzheimers disease therapy.

Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery.

ABSTRACT The delivery of large cargos of diameter above 15 nm for biomedical applications has proved challenging since it requires biocompatible, stably‐loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica‐iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60 nm in diameter and post‐functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP‐Ferritin, ˜534 kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica‐iron oxide nanovectors, which is thus a promising for biomedical applications. Graphical abstract Figure. No caption available.

Optimizing indomethacin-loaded chitosan nanoparticle size, encapsulation, and release using Box–Behnken experimental design

Indomethacin chitosan nanoparticles (NPs) were developed by ionotropic gelation and optimized by concentrations of chitosan and tripolyphosphate (TPP) and stirring time by 3-factor 3-level Box-Behnken experimental design. Optimal concentration of chitosan (A) and TPP (B) were found 0.6mg/mL and 0.4mg/mL with 120min stirring time (C), with applied constraints of minimizing particle size (R1) and maximizing encapsulation efficiency (R2) and drug release (R3). Based on obtained 3D response surface plots, factors A, B and C were found to give synergistic effect on R1, while factor A has a negative impact on R2 and R3. Interaction of AB was negative on R1 and R2 but positive on R3. The factor AC was having synergistic effect on R1 and on R3, while the same combination had a negative effect on R2. The interaction BC was positive on the all responses. NPs were found in the size range of 321-675nm with zeta potentials (+25 to +32mV) after 6 months storage. Encapsulation, drug release, and content were in the range of 56-79%, 48-73% and 98-99%, respectively. In vitro drug release data were fitted in different kinetic models and pattern of drug release followed Higuchi-matrix type.

Chemical specificity in REDOX-responsive materials: the diverse effects of different Reactive Oxygen Species (ROS) on polysulfide nanoparticles

REDOX responsive (nano)materials typically exhibit chemical changes in response to the presence and concentration of oxidants/reductants. Due to the complexity of biological environments, it is critical to ascertain whether the chemical response may depend on the chemical details of the stimulus, in addition to its REDOX potential, and whether chemically different responses can determine a different overall performance of the material. Here, we have used oxidation-sensitive materials, although these considerations can be extended also to reducible ones. In particular, we have used poly(propylene sulfide) (PPS) nanoparticles coated with a PEGylated emulsifier (Pluronic F127); inter alia, we here present also an improved preparative method. The nanoparticles were exposed to two Reactive Oxygen Species (ROS) typically encountered in inflammatory reactions, hydrogen peroxide (H2O2) and hypochlorite (ClO−); their response was evaluated with a variety of techniques, including diffusion NMR spectroscopy that allowed to separately characterize the chemically different colloidal species produced. The two oxidants triggered a different chemical response: H2O2 converted sulfides to sulfoxides, while ClO− partially oxidized them further to sulfones. The different chemistry correlated to a different material response: H2O2 increased the polarity of the nanoparticles, causing them to swell in water and to release the surface PEGylated emulsifier; the uncoated oxidized particles still exhibited very low toxicity. On the contrary, ClO− rapidly converted the nanoparticles into water-soluble, depolymerized fragments with a significantly higher toxicity. The take-home message is that it is more correct to discuss ‘smart’ materials in terms of an environmentally specific response to (REDOX) stimuli. Far from being a problem, this could open the way to more sophisticated and precisely targeted applications.

Hyaluronic Acid Coated Chitosan Nanoparticles Reduced the Immunogenicity of the Formed Protein Corona

Studying the interactions of nanoparticles (NPs) with serum proteins is necessary for the rational development of nanocarriers. Optimum surface chemistry is a key consideration to modulate the formation of the serum protein corona (PC) and the resultant immune response. We investigated the constituent of the PC formed by hyaluronic acid-coated chitosan NPs (HA-CS NPs). Non-decorated chitosan NPs (CS NPs) and alginate-coated chitosan NPs (Alg-CS NPs) were utilized as controls. Results show that HA surface modifications significantly reduced protein adsorption relative to controls. Gene Ontology analysis demonstrates that HA-CS NPs were the least immunogenic nanocarriers. Indeed, less inflammatory proteins were adsorbed onto HA-CS NPs as opposed to CS and Alg-CS NPs. Interestingly, HA-CS NPs differentially adsorbed two unique anti-inflammatory proteins (ITIH4 and AGP), which were absent from the PC of both controls. On the other hand, CS and Alg-CS NPs selectively adsorbed a proinflammatory protein (Clusterin) that was not found on the surfaces of HA-CS NPs. While further studies are needed to investigate abilities of the PCs of only ITIH4 and AGP to modulate the interaction of NPs with the host immune system, our results suggest that this proof-of-concept could potentially be utilized to reduce the immunogenicity of a wide range of nanomaterials.

Effect of cryoprotection on particle size stability and preservation of chitosan nanoparticles with and without hyaluronate or alginate coating

The aim of the present study was to determine the effect of different cryoprotectants and their concentration on the physicochemical characteristics of chitosan nanoparticles (CS-NPs). The effect of coating of CS-NPs with hyaluronic acid (HA) and alginic acid (ALG) before and after lyophilization was also evaluated. The ionic gelation method was used for the preparation of NPs and six different types of cryoprotectants (sucrose, glucose, trehalose, mannitol, polyethylene glycol-2000, and polyethylene glycol-10,000) were investigated at 5%, 10%, 20%, and 50% concentration levels. Coating of CS-NPs with HA and their protection with high amount of cryoprotectants indicated better particle size stability. Samples that were lyophilized without cryoprotectants resulted in an increase in average size due to high agglomeration. All cryoprotectants with varying amount provided some sort of size stability for the NPs except for the PEG-10,000 which had no protective effect at higher concentrations. Sucrose and trehalose sugars were found to have the highest protective effect with HA coated and uncoated CS-NPs. In conclusion, using cryoprotectants along with surface coating, the CS-NPs could achieve the desired physicochemical characteristics for a prolonged duration.