Pablo Hervella

Organic Reactivity in Aot-Stabilized Microemulsions

Microemulsions are highly versatile reaction media, which currently find many applications. In this review, we shall describe recent trends in the use of microemulsions as organic reaction media, and present models for their functioning, in particular the pseudophase model. This model allows a quantitative explanation of organic reactivity in these microheterogeneous media.

Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative

The focus of this study is to disclose a new delivery carrier intended to improve the pharmacokinetic characteristics of the anticancer drug plitidepsin and to favor its accumulation within the tumor. These nanocarriers named as nanocapsules, consist of an oily core surrounded by a highly PEGylated polyglutamic acid (PGA-PEG) shell loaded with plitidepsin. They showed a size of around 190 nm, a zeta potential of -24 mV and were able to encapsulate a high percentage (85%) of plitidepsin. In vivo studies, following intravenous injection in healthy mice, indicated that the encapsulation of the drug within PGA-PEG nanocapsules led to an important increase in its area under the curve (AUC) which is related to the important decrease of the clearance, as compared to the values observed for the drug dissolved in a Cremophor(®) EL solution. This improvement of the pharmacokinetic profile of the encapsulated plitidepsin was accompanied by a high increase (2.5-fold) of the maximum tolerated dose (MTD) in comparison to that of plitidepsin Cremophor(®) EL solution. The efficacy study performed in a xenograft tumor mice model evidenced the capacity of PGA-PEG nanocapsules to significantly reduce tumor growth. These promising results highlight the potential of PGA-PEG nanocapsules as an effective drug delivery system for cancer therapy.

Bottom up design of nanoparticles for anti-cancer diapeutics: "put the drug in the cancer's food".

Abstract The story starts in Basel at CLINAM in 2013, when I asked Pieter about making nanoparticles and he advised me to “try this solvent-exchange method we have developed for making limit sized particles”. We are particularly interested in what are “limit size materials” because we want to test the feasibility of an idea: could we design, make, develop, and test the concept for treating metastatic cancer by, “Putting the Drug in the Cancer’s Food? “Limit size” is the size of the cancer‘s food, ? the common Low Density Lipoprotein, (LDL) ~20 nm diameter. In this contribution to Pieter’s LTAA we focus on the “bottom” (nucleation) and the “up” (growth) of “bottom-up design” as it applies to homogeneous nucleation of especially, hydrophobic drugs and the 8 physico-chemical stages and associated parameters that determine the initial size, and any subsequent coarsening, of a nanoparticle suspension. We show that, when made by the rapid solvent-exchange method, the same sized particles can be obtained without phospholipid. Furthermore, the obtained size follows the predictions of classic nucleation theory when the appropriate values for the parameters (surface tension and supersaturation) at nucleation are included. Calculations on dissolution time for nanoparticles reveal that a typical fewmicromolar-solubility, hydrophobic, anti-cancer drug (like Lapatinib, Niclosamide, Abiraterone, and Fulvestrant) of 500 nm diameter would take between 3?7 s to dissolve in an infinite sink like the blood stream; and a 50 nm particle would dissolve in less than a second! And so the nanoparticle design requires a highly water-insoluble drug, and a tight, encapsulating, impermeable lipid:cholesterol monolayer. While the “Y” junction can be used to mix an ethanolic solution with anti-solvent, we find that a “no-junction” can give equally good results. A series of nanoparticles (DiI-fluorescently labeled Triolein-cored and drug-cored nanoparticles of Orlistat) were then tested in well-characterized cell lines for uptake and efficacy as well as a PET-imageable nanoparticle in initial PET-imaging studies in animals for EPR uptake and tumor detection. We show that, while free-drug cannot be optimally administered in vivo, a nanoparticle formulation of orlistat could in principle represent a stable parenteral delivery system. The article ends with a brief discussion of what we see as the way forward in Individualized Medicine from the Diagnostic-Therapeutic (“Diapeutic”) side, requiring 18FDG detection of metastatic lesions, functional imaging of a protein target (e.g. Fatty Acid Synthase) using 11C acetate, then a PET (or other)-imageable nanoparticle to demonstrate EPR accumulation, and then the administration of the pure-drug nanoparticle taken in by the most aggressive cancer cells in the perivascular space, as they would their “food”.

Inhibition of cholesterol transport in an intestine cell model by pine-derived phytosterols

We have quantified the inhibition of intestinal cholesterol transport by pine-derived phytosterols using an HT29-MTX intestine cell model that forms a mucus layer similar to that in the intestine. An artificial intestinal fluid consisting of digested fat, bile salt, cholesterol, and phytosterols was formulated in order to mimic the conditions in the intestine. The apparent permeability coefficient (Papp) of the positive control, i.e., 0.1mM of cholesterol solubilized in the artificial intestine fluid, was found to be 0.33 (±0.17)×10-6cm/s. When 0.1mM β-sitosterol was solubilized alongside, Papp was effectively zero, corresponding to a total inhibition of cholesterol transport. A similar strong inhibition was found when commercial pine-derived phytosterols, PinVita™ FSP DuPont, were co-solubilized with cholesterol in the dietary model micelles, leading to Papp=0.06 (±0.06)×10-6cm/s, i.e., 5.5 times lower than the cholesterol positive control. Additionally, the effect of potential oral administration formulations generated by the pine-derived phytosterols was also characterized. The formulations were produced as a liquid formulation of the cholesterol-containing artificial intestine fluid. Six liquid formulations were tested of which four displayed a Papp in the range of 0-0.09×10-6cm/s. The remaining two formulations did not show any inhibition effect on cholesterol transport and even enhanced cholesterol transport. It was furthermore observed that the phytosterols were found in the collected intestine cells but not transported to the basolateral region in the intestinal cell model system.

PEGylated Lipid Nanocapsules with Improved Drug Encapsulation and Controlled Release Properties

Drugs with poor lipid and water solubility are some of the most challenging to formulate in nanocarriers, typically resulting in low encapsulation efficiencies and uncontrolled release profiles. PEGylated nanocapsules (PEG-NC) are known for their amenability to diverse modifications that allow the formation of domains with different physicochemical properties, an interesting feature to address a drug encapsulation problem. We explored this problem by encapsulating in PEG-NC the promising anticancer drug candidate F10320GD1, used herein as a model for compounds with such characteristics. The nanocarriers were prepared from Miglyol(®), lecithin and PEG-sterate through a solvent displacement technique. The resulting system was a homogeneous suspension of particles with size around 200 nm. F10320GD1 encapsulation was found to be very poor (<15%) if PEG-NC were prepared using water as continuous phase; but we were able to improve this value to 85% by fixing the pH of the continuous phase to 9. Interestingly, this modification also improved the controlled release properties and the chemical stability of the formulation during storage. These differences in pharmaceutical properties together with physicochemical data suggest that the pH of the continuous phase used for PEG-NC preparation can modify drug allocation, from the external shell towards the inner lipid core of the nanocapsules. Finally, we tested the bioactivity of the drug-loaded PEG-NC in several tumor cell lines, and also in endothelial cells. The results indicated that drug encapsulation led to an improvement on drug cytotoxicity in tumor cells, but not in non-tumor endothelial cells. Altogether, the data confirms that PEG-NC show adequate delivery properties for F10320GD1, and underlines its possible utility as an anticancer therapy.

Application of the pseudophase ion-exchange model to reactivity in quaternary water in oil microemulsions

The nucleophilic attack of a bromide ion on benzyl chloride (BCl) and the acid denitrosation of N-methyl-N-nitroso-para-toluenesulfonamide (MNTS) in quaternary microemulsions of TTABr and SDS containing 1-hexanol as cosurfactant were studied. The experimental results were examined in the light of a kinetic model which allows the quaternary microemulsion to be reduced to a pseudo-ternary microemulsion, by considering the cosurfactant partition between the continuous medium and the interface of the microemulsion to be determined by Shulman titration. The incorporation of the alcohol into the interface alters the interfacial volume to be used in constructing the kinetic model. The second step of the process involves determining how the reactants partition among the different microenvironments of the pseudo-ternary system, the interface being the sole reactive region in the microemulsion. Accounting for the results obtained in the acid denitrosation of MNTS required using the ion-exchange formalism. Based on the results, the product of the fraction of neutralized charge at the interface and the rate constant is independent of the water content of the system. This suggests that the degree of dissociation of the surfactant at the interface does not depend on the water content of the microemulsion. Under the assumption of a surfactant fraction of neutralized charge of β = 0.8, we calculated the rate constants at the interface for both processes. Such rate constants were smaller than in the aqueous medium and consistent with the effects found in nitroso group transfer reactions in microemulsions.

Real-Time Visualization of the Precipitation and Phase Behavior of Octaethylporphyrin in Lipid Microparticles

The material properties of micro- and nanoparticles are fundamental for their bulk properties in suspension, like their stability and encapsulation efficiency. A particularly interesting system with potential biomedical applications is the encapsulation of hydrophobic porphyrins into lipid particles and their use as metal atom chelators, where retention and stability are keys for the design process. The overall goal here was to study the solubility, phase behavior, and mixing of octaethylporphyrin (OEP) and OEP-Cu chelates with 2 core materials, triolein (TO) and cholesteryl acetate, as single microparticles. We employed a real-time, single-particle microscopic technique based on micropipette injection to characterize the behavior of these materials and their mixtures upon solvent loss and precipitation. A clear phase separation was observed between the triolein liquid core and porphyrin microcrystals, and the ternary phase diagram of the droplet compositions and onsets of phase separation over solvent dissolution was built. On the contrary, cholesteryl acetate and OEP-Cu coprecipitated by solvent dissolution, preventing porphyrin crystallization even for very high supersaturations. This type of real-time, single-particle characterization is expected to offer important information about the formulation of other hydrophobic compounds of interest, where finding the proper encapsulation environment is a key step for their retention and stability.

Data for the size of cholesterol-fat micelles as a function of bile salt concentration and the physico-chemical properties of six liquid experimental pine-derived phytosterol formulations in a cholesterol-containing artificial intestine fluid

The data in this paper are additional information to the research article entiltled “Inhibition of cholesterol transport in an intestine cell model by pine-derived phytosterols” (Yi et al.,2016) [1]. The data derived from the measurement on six liquid formulations of commercial pine-derived phytosterol (CPP) by dynamic light scattering. The data cover micelle size and the zeta-potential for formulations with cholesterol including monoglyceride, oleic acid, and bile salt. The data demonstrate the critical effect of the bile salt concentration on the size of cholesterol-digested fat micelles.

Chelation, formulation, encapsulation, retention, and in vivo biodistribution of hydrophobic nanoparticles labelled with 57Co-porphyrin: Oleylamine ensures stable chelation of cobalt in nanoparticles that accumulate in tumors

Background and motivation: While small molecules can be used in cancer diagnosis there is a need for imageable diagnostic NanoParticles (NPs) that act as surrogates for the therapeutic NPs. Many NPs are composed of hydrophobic materials so the challenge is to formulate hydrophobic imaging agents. To develop individualized medical treatments based on NP, a first step should be the selection of patients who are likely responders to the treatment as judged by imaging tumor accumulation of NPs. This requires NPs with the same size and structure as the subsequent therapeutic NPs but labelled with a long‐lived radionuclide. Cobalt isotopes are good candidates for NP labelling since 55Co has half‐life of 17.5h and positron energy of 570keV while 57Co (t1/2 271.6 d) is an isotope suited for preclinical single photon emission tomography (SPECT) to visualize biodistribution and pharmacokinetics of NPs. We used the hydrophobic octaethyl porphyrin (OEP) to chelate cobalt and to encapsulate it inside hydrophobic liquid NPs (LNPs). We hypothesized that at least two additional hydrophobic axial ligands (oleylamine, OA) must be provided to the OEP‐Co complex in order to encapsulate and retain Co inside LNP. Results: 1. Cobalt chelation by OEP and OA. The association constant of cobalt to OEP was 2.49×105 M−1 and the formation of the hexacoordinate complex OEP‐Co‐4OA was measured by spectroscopy. 2. NP formulation and characterization: LNPs were prepared by the fast ethanol injection method and were composed of a liquid core (triolein) surrounded by a lipid monolayer (DSPC:Cholesterol:DSPE‐PEG2000). The size of the LNPs loaded with the cobalt complex was 40±5nm, 3. Encapsulation of OEP‐Co‐OA: The loading capacity of OEP‐Co‐OA in LNP was 5mol%. 4. Retention of OEP‐57Co‐4OA complex in the LNPs: the positive effect of the OA ligands was demonstrated on the stability of the OEP‐57Co‐4OA complex, providing a half‐life for retention in PBS of 170h (7days) while in the absence of the axial OA ligands was only 22h. 5 Biodistribution Study: the in vivo biodistribution of LNP was studied in AR42J pancreatic tumor‐bearing mice. The estimated half‐life of LNPs in blood was about 7.2h. Remarkably, the accumulation of LNPs in the tumor was as high as 9.4% ID/g 24h after injection with a doubling time for tumor accumulation of 3.22h. The most important result was that the nanoparticles could indeed accumulate in the AR42J tumors up to levels greater than those of other NPs previously measured in the same tumor model, and at about half the values reported for the molecular agent 57Co‐DOTATATE. Conclusions: The additional hydrophobic chelator OA was indeed needed to obtain a stable octahedral OEP‐Co‐4OA. Cobalt was actually well‐retained inside LNP in the OEP‐Co‐4OA complex. The method described in the present work for the core‐labelling of LNPs with cobalt is now ready for labeling of NPs with 55Co, or indeed other hexadentate radionuclides of interest for preclinical in vivo PET‐imaging and radio‐therapeutics.

Formulation and in vivo biodistribution of 57Co-porphyrin-labelled hydrophobic liquid nanoparticles

European Journal of Nuclear Medicine and Molecular Imaging Volume 44, Supplement 2 10.1007/s00259-017-3822-1 This supplement was not sponsored by outside commercial interests. It was funded entirely by the association’s own resources DOI 10.1007/s00259-017-3822-1 S119 Eur J Nucl Med Mol Imaging (2017) 44 (Suppl 2):S119–S956