Overcoming Solubility Challenges: Liposomal isoCoQ-Carbazole as a Promising Anti-Tumor Agent for Inoperable and Radiation-Insensitive cancers.

This study evaluated the potential of isoCoQ-Carbazole, a diheterocyclic analog of isoCA-4, as an anti-tumor agent. To overcome its low aqueous solubility, liposomes were developed as a delivery system for the compound. In vitro experiments showed that loaded liposomes exhibited similar activity to the free form on multiple human tumor cell lines. In vivo experiments using a palliative intratumoral injection chemotherapy approach further demonstrated that isoCoQ-Carbazole loaded liposomes significantly reduced tumor growth in a CA-4-resistant HT29 cell model, without inducing any observable toxicity or weight loss in the treated mice. These findings suggest that liposomal isoCoQ-Carbazole may hold promise as a potential therapeutic agent for the treatment of inoperable, radiation-insensitive cancers.

Degradable polyisoprene by radical ring-opening polymerization and application to polymer prodrug nanoparticles.

Radical ring-opening polymerization (rROP) has received renewed attention to incorporate cleavable linkages into the backbones of vinyl polymers, especially from cyclic ketene acetals (CKAs). Among the monomers that hardly copolymerize with CKAs are (1,3)-dienes such as isoprene (I). This is unfortunate since synthetic polyisoprene (PI) and its derivatives are the materials of choice for many applications, in particular as elastomers in the automotive, sport, footwear, and medical industries, but also in nanomedicine. Thionolactones have been recently proposed as a new class of rROP-compatible monomers for insertion of thioester units in the main chain. Herein, we report the synthesis of degradable PI by rROP via the copolymerization of I and dibenzo[c,e]oxepane-5-thione (DOT). Free-radical polymerization as well as two reversible deactivation radical polymerization techniques were successfully used for the synthesis of (well-defined) P(I-co-DOT) copolymers with adjustable molecular weights and DOT contents (2.7-9.7 mol%). Reactivity ratios of r DOT = 4.29 and r I = 0.14 were determined, suggesting preferential incorporation of DOT in comparison to I. The resulting P(I-co-DOT) copolymers were successfully degraded (from -47% to -84% decrease in M n) under basic conditions. As a proof of concept, the P(I-co-DOT) copolymers were formulated into stable and narrowly dispersed nanoparticles, showing similar cytocompatibility on J774.A1 and HUVEC cells compared to their PI counterparts. Furthermore, Gem-P(I-co-DOT) prodrug nanoparticles were synthesized by the "drug-initiated" method and exhibited significant cytotoxicity on A549 cancer cells. P(I-co-DOT) and Gem-P(I-co-DOT) nanoparticles were degraded under basic/oxidative conditions by bleach and under physiological conditions in the presence of cysteine or glutathione.

Vinyl copolymers with faster hydrolytic degradation than aliphatic polyesters and tunable upper critical solution temperatures.

Vinyl polymers are the focus of intensive research due to their ease of synthesis and the possibility of making well-defined, functional materials. However, their non-degradability leads to environmental problems and limits their use in biomedical applications, allowing aliphatic polyesters to still be considered as the gold standards. Radical ring-opening polymerization of cyclic ketene acetals is considered the most promising approach to impart degradability to vinyl polymers. However, these materials still exhibit poor hydrolytic degradation and thus cannot yet compete with traditional polyesters. Here we show that a simple copolymerization system based on acrylamide and cyclic ketene acetals leads to well-defined and cytocompatible copolymers with faster hydrolytic degradation than that of polylactide and poly(lactide-co-glycolide). Moreover, by changing the nature of the cyclic ketene acetal, the copolymers can be either water-soluble or can exhibit tunable upper critical solution temperatures relevant for mild hyperthermia-triggered drug release. Amphiphilic diblock copolymers deriving from this system can also be formulated into degradable, thermosensitive nanoparticles by an all-water nanoprecipitation process.

Gemcitabine Lipid Prodrugs: The Key Role of the Lipid Moiety on the Self-Assembly into Nanoparticles.

A small library of amphiphilic prodrugs has been synthesized by conjugation of gemcitabine (Gem) (a hydrophilic nucleoside analogue) to a series of lipid moieties and investigated for their capacity to spontaneously self-assemble into nanosized objects by simple nanoprecipitation. Four of these conjugates formed stable nanoparticles (NPs), while with the others, immediate aggregation occurred, whatever the tested experimental conditions. Whether such capacity could have been predicted based on the prodrug physicochemical features was a matter of question. Among various parameters, the hydrophilic-lipophilic balance (HLB) value seemed to hold a predictive character. Indeed, we identified a threshold value which well correlated with the tendency (or not) of the synthesized prodrugs to form stable nanoparticles. Such a hypothesis was further confirmed by broadening the analysis to Gem and other nucleoside prodrugs already described in the literature. We also observed that, in the case of Gem prodrugs, the lipid moiety affected not only the colloidal properties but also the in vitro anticancer efficacy of the resulting nanoparticles. Overall, this study provides a useful demonstration of the predictive potential of the HLB value for lipid prodrug NP formulation and highlights the need of their opportune in vitro screening, as optimal drug loading does not always translate in an efficient biological activity.

Elongated self-assembled nanocarriers: From molecular organization to therapeutic applications.

Self-assembled cylindrical aggregates made of amphiphilic molecules emerged almost 40 years ago. Due to their length up to micrometers, those particles display original physico-chemical properties such as important flexibility and, for concentrated samples, a high viscoelasticity making them suitable for a wide range of industrial applications. However, a quarter of century was needed to successfully take advantage of those improvements towards therapeutic purposes. Since then, a wide diversity of biocompatible materials such as polymers, lipids or peptides, have been developed to design self-assembling elongated drug nanocarriers, suitable for therapeutic or diagnostic applications. More recently, the investigation of the main forces driving the unidirectional growth of these nanodevices allowed a translation toward the formation of pure nanodrugs to avoid the use of unnecessary side materials and the possible toxicity concerns associated.

Squalene-based multidrug nanoparticles for improved mitigation of uncontrolled inflammation in rodents.

Uncontrolled inflammatory processes are at the root of numerous pathologies. Most recently, studies on confirmed COVID-19 cases have suggested that mortality might be due to virally induced hyperinflammation. Uncontrolled pro-inflammatory states are often driven by continuous positive feedback loops between pro-inflammatory signaling and oxidative stress, which cannot be resolved in a targeted manner. Here, we report on the development of multidrug nanoparticles for the mitigation of uncontrolled inflammation. The nanoparticles are made by conjugating squalene, a natural lipid, to adenosine, an endogenous immunomodulator, and then encapsulating α-tocopherol, as antioxidant. This resulted in high drug loading, biocompatible, multidrug nanoparticles. By exploiting the endothelial dysfunction at sites of acute inflammation, these multidrug nanoparticles delivered the therapeutic agents in a targeted manner, conferring survival advantage to treated animals in models of endotoxemia. Selectively delivering adenosine and antioxidants together could serve as a novel therapeutic approach for safe treatment of acute paradoxal inflammation.

A Self-Assembling NHC-Pd-Loaded Calixarene as a Potent Catalyst for the Suzuki-Miyaura Cross-Coupling Reaction in Water.

Nanoformulated calix[8]arenes functionalized with N-heterocyclic carbene (NHC)-palladium complexes were found to be efficient nano-reactors for Suzuki-Miyaura cross-coupling reactions of water soluble iodo- and bromoaryl compounds with cyclic triol arylborates at low temperature in water without any organic co-solvent. Combined with an improved one-step synthesis of triol arylborates from boronic acid, this remarkably efficient new tool provided a variety of 4'-arylated phenylalanines and tyrosines in good yields at low catalyst loading with a wide functional group tolerance.

Stacking as a Key Property for Creating Nanoparticles with Tunable Shape: The Case of Squalenoyl-Doxorubicin.

The development of elongated nanoparticles for drug delivery is of growing interest in recent years, due to longer blood circulation and improved efficacy compared to spherical counterparts. Squalenoyl-doxorubicin (SQ-Dox) conjugate was previously shown to form elongated nanoparticles with improved therapeutic efficacy and decreased toxicity compared to free doxorubicin. By using experimental and computational techniques, we demonstrate here that the specific physical properties of SQ-Dox, which include stacking and electrostatic interactions of doxorubicin as well as hydrophobic interactions of squalene, are involved in the formation of nanoassemblies with diverse elongated structures. We show that SQ-Dox bioconjugate concentration, ionic strength, and anion nature can be used to modulate the shape and stiffness of SQ-Dox nanoparticles. As those parameters are involved in nanoparticle behavior in biological media, these findings could bring interesting opportunities for drug delivery and serve as an example for the design of original nanodrugs with stacking properties tuned for particular clinical purposes.

Drug-Initiated Synthesis of Heterotelechelic Polymer Prodrug Nanoparticles for in Vivo Imaging and Cancer Cell Targeting.

" Drug-initiated" nitroxide-mediated synthesis of two well-defined, heterotelechelic polymer prodrugs ( Mn = 1960-5260 g·mol-1, D = 1.31-1.37) was performed by using the newly developed nitroxide exchange reaction. These polymers comprised, at the chain end, gemcitabine (Gem) as anticancer drug and either cyanine 7.5 (Cy7.5) as a near-infrared (NIR) dye suitable for in vivo imaging or biotin (Biot) for cancer cell targeting. These materials were co-nanoprecipitated into fluorescently labeled polymer prodrug nanoparticles of average diameter in the 100-180 nm range with narrow particle size distribution and variable surface amounts of biotin. Nanoparticles containing 15 wt % biotinylated polymer showed superior uptake and the highest cytotoxicity in vitro on A549 human lung cancer cells. In vivo, on A549 tumor bearing mice, biotinylated nanoparticles showed significantly higher efficacy than free Gem and maintained the same anticancer activity than nontargeted nanoparticles without inducing prohibitive body weight loss. Biotinylated polymer prodrug nanoparticles did not result in an improved anticancer activity or significant increase in tumor accumulation, which may be the result of a nonoptimal biotin surface display and/or insufficient affinity toward the target. They however displayed delayed liver accumulation compared to nonbiotinylated counterparts, suggesting the premise of a stealth property likely due to the hydrophilic tetraethylene glycol-Biot positioned at the nanoparticle surface. This work showed for the first time the applicability of this simple construction method to in vivo imaging and cancer cell targeting and might stimulate the design of new functional materials for biomedical applications.

Squalene-Adenosine Nanoparticles: Ligands of Adenosine Receptors or Adenosine Prodrug?

Adenosine receptors (ARs) represent key drug targets in many human pathologies, including cardiovascular, neurologic, and inflammatory diseases. To overcome the very rapid metabolization of adenosine, metabolically stable AR agonists and antagonists were developed. However, few of these molecules have reached the market due to efficacy and safety issues. Conjugation of adenosine to squalene to form squalene-adenosine (SQAd) nanoparticles (NPs) dramatically improved the pharmacological efficacy of adenosine, especially for neuroprotection in stroke and spinal cord injury. However, the mechanism by which SQAd NPs displayed therapeutic activity remained totally unknown. In the present study, two hypotheses were discussed: 1) SQAd bioconjugates, which constitute the NP building blocks, act directly as AR ligands; or 2) adenosine, once released from intracellularly processed SQAd NPs, interacts with these receptors. The first hypothesis was rejected, using radioligand displacement assays, as no binding to human ARs was detected, up to 100 µM SQAd, in the presence of plasma. Hence, the second hypothesis was examined. SQAd NPs uptake by HepG2 cells, which was followed using radioactive and fluorescence tagging, was found to be independent of equilibrative nucleoside transporters but rather mediated by low-density lipoprotein receptors. Interestingly, it was observed that after cell internalization, SQAd NPs operated as an intracellular reservoir of adenosine, followed by a sustained release of the nucleoside in the extracellular medium. This resulted in a final paracrine-like activation of the AR pathway, evidenced by fluctuations of the second messenger cAMP. This deeper understanding of the SQAd NPs mechanism of action provides a strong rational for extending the pharmaceutical use of this nanoformulation.

Heterotelechelic polymer prodrug nanoparticles: Adaptability to different drug combinations and influence of the dual functionalization on the cytotoxicity.

Well-defined, heterotelechelic polymer prodrugs for combination therapy were synthesized by using a combination of the "drug-initiated" nitroxide-mediated polymerization from a gemcitabine-alkoxyamine initiator and the nitroxide exchange reaction using TEMPO-bearing drugs to end-cap the drug-polymer chain-end by a second drug. This methodology was successfully applied to two different clinically relevant combinations, gemcitabine/doxorubicin (Gem/Dox) and gemcitabine/lapatinib (Gem/Lap), showing a certain degree of universality of the synthetic methodology. It also represented the first nanocarrier for the co-delivery of Gem and Lap ever reported. Well-controlled, low molar mass heterotelechelic polymers (Mn = 2100-4090 g.mol-1, Ð = 1.18-1.38) with ~1:1 drug ratios and high overall drug loadings up to 40 wt% were obtained. They were formulated into nanoparticles by nanoprecipitation and exhibited average diameters in the 34-154 nm range, with narrow particle size distributions (PSD = 0.01-0.22) and excellent colloidal stability over time. Their biological evaluation in terms of drug release and cytotoxicity was performed and compared to that of different monofunctional polymer prodrug formulations. We showed that heterobifunctional polymer prodrugs induced cytotoxicity to MCF-7 cells, with IC50 values in the 120-300 nM range depending on the combination tested. Interestingly, whereas Gem/Dox combination did not lead to noticeable improvement over monofunctional therapies, co-nanoprecipitation of Gem/Lap prodrugs led to synergistic effect.

Conjugation of squalene to gemcitabine as unique approach exploiting endogenous lipoproteins for drug delivery.

Once introduced in the organism, the interaction of nanoparticles with various biomolecules strongly impacts their fate. Here we show that nanoparticles made of the squalene derivative of gemcitabine (SQGem) interact with lipoproteins (LPs), indirectly enabling the targeting of cancer cells with high LP receptors expression. In vitro and in vivo experiments reveal preeminent affinity of the squalene-gemcitabine bioconjugates towards LP particles with the highest cholesterol content and in silico simulations further display their incorporation into the hydrophobic core of LPs. To the best of our knowledge, the use of squalene to induce drug insertion into LPs for indirect cancer cell targeting is a novel concept in drug delivery. Interestingly, not only SQGem but also other squalene derivatives interact similarly with lipoproteins while such interaction is not observed with liposomes. The conjugation to squalene represents a versatile platform that would enable efficient drug delivery by simply exploiting endogenous lipoproteins.

A Squalene-Based Nanomedicine for Oral Treatment of Colon Cancer.

Nanotechnology offers many possibilities to improve drug treatments, including with regard to drug pharmacology. The current study reports a simple approach to improve cisplatin efficacy in the treatment of colon cancer through the creation of orally administered squalenoylated nanoparticles loaded with cisplatin (SQ-CDDP NP). Cytotoxic effects of SQ-CDDP NP were assessed in human colonic cells and in mouse models of intestinal cancer. In cell culture, SQ-CDDP NP exhibited at least 10-fold greater cytotoxic potency compared with uncomplexed cisplatin, reflecting an enhancement in intracellular accumulation and DNA platination. Mechanistic investigations showed that SQ-CDDP NP stimulated ROS production, expression of heavy metal-inducible and stress-inducible genes, stress kinase cascades, and apoptosis. In ApcMin/+ mice, a model of intestinal tumorigenesis, oral administration of SQ-CDDP NP curtailed spontaneous tumor formation and azoxymethane-induced colon carcinogenesis with no apparent evidence of tissue toxicity. Our results offer preclinical validation of a nanocarrier formulation that can safely improve chemotherapeutic efficacy, address risks of drug resistance, and improve patient compliance by enabling oral administration. Cancer Res; 77(11); 2964-75. ©2017 AACR.

In vitro investigation of multidrug nanoparticles for combined therapy with gemcitabine and a tyrosine kinase inhibitor: Together is not better.

Combined therapy with gemcitabine and tyrosine-kinase inhibitors (i.e., sunitinib) has already demonstrated important benefits in pancreatic cancer treatment. Further therapeutic advantage could be achieved by their co-loading in a single nanoscale system, which enables (i) the co-existence of drugs with different mechanisms of action and pharmacokinetic profiles and (ii) the fine tuning of their release rate overcoming the rapid clearance often observed with free drugs. In this context, the already validated squalenoylation approach has been applied to the design of a multidrug nanoparticle (NP) made by co-self-assembly of the squalene-based prodrugs of gemcitabine (SQGem) and sunitinib (SQSun). We hypothesized that co-delivering of SQGem and SQSun in a single nanoparticle was capable to increase their cytotoxicity on MIA PaCa-2 pancreatic cancer cells compared to the monodrug NPs. Nevertheless, multidrug NPs (i.e., SQGem/SQSun NPs) were as efficient as the physical mixture of the individual monodrug NPs (SQGem NPs + SQSun NPs) thus suggesting that the cytotoxicity raised from the exposure of the cells simultaneously to the two bioconjugates rather than to their original loading into a single or two different nanoparticles. To be noted that the lack of differences in static 2D cultures does not exclude a different behavior in dynamic conditions in vivo.

An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance.

The "Golden era" of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55-63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.

Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis.

Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury.

Gemcitabine-based therapy for pancreatic cancer using the squalenoyl nucleoside monophosphate nanoassemblies.

Gemcitabine is currently the most effective agent against advanced pancreatic cancer. However, the major therapeutic hurdles using gemcitabine include rapid inactivation by blood deaminases and fast development of cell chemoresistance, induced by down-regulation of deoxycytidine kinase or nucleoside transporters. To overcome the above drawbacks we designed recently a novel nanomedicine strategy based on squalenoyl prodrug of 5'-monophosphate gemcitabine (SQdFdC-MP). This amphiphilic conjugate self-organized in water into unilamellar vesicles with a mean diameter of 100 nm. In this study the antitumor efficacy of SQdFdC-MP nanoassemblies (NAs) on chemoresistant and chemosensitive pancreatic adenocarcinoma models have been investigated. Cell viability assays showed that SQdFdC-MP NAs displayed higher antiproliferative and cytotoxic effects, particularly in chemoresistant pancreatic tumor cells. In in vivo studies, SQdFdC-MP NAs decreased significantly the growth (∼70%) of human MiaPaCa2 xenografts, also preventing tumor cell invasion, whereas native dFdC did not display any anticancer activity when tumor growth inhibition was only 35% with SQdFdC NAs. These results correlated with a reduction of Ki-67 antigen and the induction of apoptosis mediated by caspase-3 activation in tumor cells. These findings demonstrated the feasibility of utilizing SQdFdC-MP NAs to make tumor cells more sensitive to gemcitabine and thus providing an efficient new therapeutic alternative for pancreatic adenocarcinoma.

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury.

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.