Squalenoyl Gemcitabine Research Articles

Combinatorial Nanomedicine Made of Squalenoyl-Gemcitabine and Edelfosine for the Treatment of Osteosarcoma.

Due to chemoresistance and a high propensity to form lung metastasis, survival rates in pediatric osteosarcoma (OS) are poor. With the aim to improve anticancer activity in pediatric OS, a multidrug nanomedicine was designed using the alkyl-lysophospholipid edelfosine (EF) co-assembled with squalenoyl–gemcitabine (SQ–Gem) to form nanoassemblies (NAs) of 50 nm. SQ–Gem/EF NAs modified the total Gem pool exposure in the blood stream in comparison with SQ–Gem NAs, which correlated with a better tolerability and a lower toxicity profile after multiple intravenous administrations in mice. For in vivo preclinical assessment in an orthotopic OS tumor model, P1.15 OS cells were intratibially injected in athymic nude mice. SQ–Gem/EF NAs considerably decreased the primary tumor growth kinetics and reduced the number of lung metastases. Our findings support the candidature of this anticancer nanomedicine as a potential pediatric OS therapy.

Synthesis of a deuterated probe for the confocal Raman microscopy imaging of squalenoyl nanomedicines.

The synthesis of ω-di-(trideuteromethyl)-trisnorsqualenic acid has been achieved from natural squalene. The synthesis features the use of a Shapiro reaction of acetone-d6 trisylhydrazone as a key step to implement the terminal isopropylidene-d6 moiety. The obtained squalenic acid-d6 has been coupled to gemcitabine to provide the deuterated analogue of squalenoyl gemcitabine, a powerful anticancer agent endowed with self-assembling properties. The Raman spectra of both deuterated and non-deuterated squalenoyl gemcitabine nanoparticles displayed significant Raman scattering signals. They revealed no differences except from the deuterium peak patterns in the silent spectral region of cells. This paves the way for label-free intracellular trafficking studies of squalenoyl nanomedicines.

Peptide conjugation: before or after nanoparticle formation?

We report herein a detailed study concerning the impact of different bioconjugation and nanoformulation strategies on the in vitro targeting ability of peptide-decorated squalenoyl gemcitabine (SQdFdC) nanoparticles (NPs). NPs have been functionalized with the CKAAKN peptide, previously identified as an efficient homing device within the pancreatic pathological microenvironment. Two approaches have been followed: (i) either the CKAAKN peptide was directly conjugated at the surface of preformed SQdFdC nanoparticles (conjugation after NP formation) or (ii) it was first reacted with a maleimide squalenoyl derivative before the resulting bioconjugate was co-nanoprecipitated with SQdFdC to form the peptide-decorated NPs (conjugation before NP formation). NPs were characterized with respect to mean diameter, zeta potential, and stability over time. Then, their specific interaction with the sFRP-4 protein was evaluated by surface plasmon resonance. Although both synthetic strategies allowed us to formulate NPs able to interact with the corresponding receptor, enhanced target binding and better specific avidity were observed with CKAAKN-NPs functionalized before NP formation. These NPs displayed the highest cell uptake and cytotoxicity in an in vitro model of human MIA Paca-2 pancreatic cancer cells.

Interaction of an amphiphilic squalenoyl prodrug of gemcitabine with cellular membranes

We have designed an amphiphilic prodrug of the anticancer agent gemcitabine (dFdC), by covalent coupling to squalene. This bioconjugate, which self-assembled into nanoparticles (NPs) in water, was previously found to display an impressive anticancer activity both in vitro and in vivo. The present study aims to investigate the impact of SQdFdC nanoparticles on cellular membranes. MTT assays showed that, in the nanomolar range, squalenoyl gemcitabine (SQdFdC) was slightly less active than dFdC on a panel of human cancer cell lines, in vitro. However, above 10 μmol L −1 SQdFdC was considerably more cytotoxic than dFdC. Contrarily to its parent drug, SQdFdC also induced cell lysis in a few hours, as evidenced by LDH release assays. Erythrocytes were used as an experimental model insensitive to the antimetabolic activity of dFdC to further investigate the putative membrane-related cytotoxic activity of SQdFdC. The bioconjugate also induced hemolysis in a time- and dose-dependent fashion, unlike squalene or dFdC, which clearly proved that SQdFdC could permeabilize cellular membranes. Structural X-ray diffraction and calorimetry studies were conducted in order to elucidate the mechanism accounting for these observations. They confirmed that SQdFdC could be transferred from NPs to phospholipid bilayers and that the insertion of the prodrug within model membranes resulted in the formation of nonlamellar structures, which are known to promote membrane leakage. As a whole, our results suggested that due to its amphiphilic nature, the cell uptake of SQdFdC resulted in its insertion into cellular membranes, which could lead to the formation of nonlamellar structures and to membrane permeation. Whether this mechanism could be the source of toxicity in vivo, however, remains to be established, since preclinical studies have clearly proven that squalenoyl gemcitabine displayed a good toxicity profile.

Interaction of Self-Assembled Squalenoyl Gemcitabine Nanoparticles with Phospholipid−Cholesterol Monolayers Mimicking a Biomembrane

Gemcitabine (dFdC or Gem) is a water-soluble cytotoxic drug, with poor cellular uptake in the absence of a nucleoside transporter. To improve its diffusion through membranes, it was modified by grafting of a squalenoyl moiety. In water, this derivative is able to form stable and monodispersed nanoparticles made of inverse hexagonal phases. The formation and interfacial properties of the squalenoyl gemcitabine (SQ-Gem) nanoparticles, and their ability to interact with phospholipid and cholesterol monolayers modeling a biomembrane, was assessed from surface tension measurements and Brewster angle microscopy. To get a better insight into the mechanisms of SQ-Gem interaction with the various lipids, the interfacial behavior of SQ-Gem and squalene was also studied by surface pressure and surface potential measurements, in the absence and in the presence of phospholipids and cholesterol. The results showed that SQ-Gem nanoparticles adsorbed at the free air/water interface and disrupted to form a monolayer. SQ-Gem molecules released from the adsorbed nanoparticles were also able to penetrate into condensed phospholipid-cholesterol mixed monolayers. The kinetics of this penetration was apparently controlled by intermolecular interactions between the drug and the adsorbed lipids. Whereas distearoylphosphatidylcholine (DSPC) hindered SQ-Gem penetration, cholesterol favored it, which could have important implications in the therapeutic field since cholesterol targeting could alter lipid raft composition and cancer cell survival.

Squalenoyl gemcitabine nanomedicine overcomes the low efficacy of gemcitabine therapy in pancreatic cancer

Development of chemoresistance and rapid inactivation of gemcitabine (Gem), the standard therapy for advanced pancreatic cancer, are responsible of the major therapeutic failures. To overcome the above drawbacks we designed a novel nanomedicine strategy for Gem nanoparticle (NP) formulation based on squalene conjugation. The purpose was to investigate the antitumor efficacy of gemcitabine-squalene (SQ-Gem) NPs on chemoresistant and chemosensitive pancreatic adenocarcinoma models. Cell viability and apoptosis assays showed that SQ-Gem NPs displayed higher antiproliferative and cytotoxic effects, particularly in chemoresistant Panc1 tumor cells. In in vivo studies, compared to native Gem, SQ-Gem NPs decreased significantly the tumor growth, prevented tumor cell invasion, and prolonged the survival time of mice bearing orthotopic pancreatic tumors. These results correlate with a greater reduction of Ki-67 and induction of apoptosis. These findings demonstrate the feasibility of utilizing SQ-Gem NPs to make tumor cells more sensitive to Gem and thus provide an efficient new therapeutic alternative for pancreatic adenocarcinoma. From the Clinical EditorPancreatic malignancies represent some of the most notoriously treatment resistant cancer varieties. This paper discusses a novel and promising nanotechnology-based treatment approach, currently at the basic science stage.

Squalene Based Nanocomposites: A New Platform for the Design of Multifunctional Pharmaceutical Theragnostics

This study reports the design of a novel theragnostic nanomedicine which combines (i) the ability to target a prodrug of gemcitabine to an experimental solid tumor under the influence of a magnetic field with (ii) the imaging of the targeted tumoral nodule. This concept is based on the inclusion of magnetite nanocrystals into nanoparticles (NPs) constructed by self-assembling molecules of the squalenoyl gemcitabine (SQgem) bioconjugate. The nanocomposites are characterized by an unusually high drug loading, a significant magnetic susceptibility, and a low burst release. When injected to the L1210 subcutaneous mice tumor model, these magnetite/SQgem NPs were magnetically guided, and they displayed considerably greater anticancer activity than the other anticancer treatments (magnetite/SQgem NPs nonmagnetically guided, SQgem NPs, or gemcitabine free in solution). The histology and immunohistochemistry investigation of the tumor biopsies clearly evidenced the therapeutic superiority of the magnetically guided nanocomposites, while Prussian blue staining confirmed their accumulation at the tumor periphery. The superior therapeutic activity and enhanced tumor accumulation has been successfully visualized using T(2)-weighted imaging in magnetic resonance imaging (MRI). This concept was further enlarged by (i) the design of squalene-based NPs containing the T(1) Gd(3+) contrast agent instead of magnetite and (ii) the application to other anticancer squalenoyls, such as, cisplatin, doxorubicin, and paclitaxel. Thus, by combining different anticancer medicines as well as contrast imaging agents in NPs, we open the door toward generic conceptual framework for cancer treatment and diagnosis. This new theragnostic nanotechnology platform is expected to have important applications in cancer therapy.

Transmembrane diffusion of gemcitabine by a nanoparticulate squalenoyl prodrug: An original drug delivery pathway

We have designed an amphiphilic prodrug of gemcitabine (dFdC) by its covalent coupling to a derivative of squalene, a natural lipid. The resulting bioconjugate self-assembled spontaneously in water as nanoparticles that displayed a promising in vivo anticancer activity. The aim of the present study was to provide further insight into the in vitro subcellular localization and on the metabolization pathway of the prodrug. Cells treated with radiolabelled squalenoyl gemcitabine (SQdFdC) were studied by differential detergent permeation, and microautography coupled to fluorescent immunolabeling and confocal microscopy. This revealed that the bioconjugate accumulated within cellular membranes, especially in those of the endoplasmic reticulum. Radio-chromatography analysis proved that SQdFdC delivered dFdC directly in the cell cytoplasm. Mass spectrometry studies confirmed that gemcitabine was then either converted into its biologically active triphosphate metabolite or exported from the cells through membrane transporters. To our knowledge, this is the first description of such an intracellular drug delivery pathway. In vitro cytotoxicity assays revealed that SQdFdC was more active than dFdC on a transporter-deficient human resistant leukemia model, which was explained by the subcellular distribution of the drugs and their metabolites. The squalenoylation drug delivery strategy might, therefore, dramatically improve the efficacy of gemcitabine on transporter-deficient resistant cancer in the clinical context.

Extracellular-protein-enhanced cellular uptake of squalenoyl gemcitabine from nanoassemblies

The mechanism by which the squalenoyl prodrug of the anticancer agent gemcitabine was transferred from its nanoassemblies (NAs) to cancer cell lines was investigated by means of in vitro cell uptake experiments. The endocytosis of the NAs was proven not to constitute the main uptake pathway of the squalenoyl gemcitabine bioconjugate (SQdFdC), which indicated that the uptake occurred by passive diffusion. On the contrary, the cell uptake was found importantly influenced by the concentration and nature of the extracellular proteins: it was very limited in protein-free media, reached a maximum at intermediate protein concentrations, and then strongly diminished at high extracellular protein concentrations. This phenomenon was demonstrated not to stem from the alteration of the colloidal stability of the NAs that could have occurred upon modification of the protein concentration. Our results indicated that SQdFdC cell uptake happened through an albumin-enhanced diffusion of molecular SQdFdC across the aqueous medium, rather than through a direct interaction between NAs and cells. Noteworthy, in our in vitro experimental conditions, lipoproteins were detrimental to the cell uptake because they trapped the available SQdFdC molecules without mediating their diffusive capture by the cells.

Anticancer Efficacy of Squalenoyl Gemcitabine Nanomedicine on 60 Human Tumor Cell Panel and on Experimental Tumor

Gemcitabine (2',2'-difluorodeoxyribofuranosylcytosine) is an anticancer nucleoside analogue active against a wide variety of solid tumors. However, following intravenous administration, this drug is rapidly inactivated by enzymatic deamination and displays a short biological half-life necessitating the administration of high doses leading also to unwanted side effects. To overcome these drawbacks and to improve the therapeutic index of gemcitabine, we have recently developed the concept of squalenoylation which consisted in the bioconjugation of gemcitabine with squalene, a natural lipid. In our preliminary studies, we have shown that this bioconjugate (SQgem) self-organized in water as nanoassemblies with considerable resistance to deamination and significantly higher anticancer activity compared with gemcitabine in an intravenously grafted tumor model in mice. To further establish the candidature of this nanomedicine for clinical trials, in this communication we have tested the preclinical efficacy of squalenoyl gemcitabine nanomedicine on several human tumor cell lines and on the subcutaneously grafted experimental L1210 murine tumor in mice. SQgem nanomedicine displayed an efficient cytotoxicity against a variety of human tumor cell lines in the 60 human tumor cell panel. In vivo, following intravenous administration, SQgem nanomedicine displayed a superior anticancer activity against subcutaneous L1210 tumor, comparatively to gemcitabine. The molecular mechanism behind the anticancer efficacy of SQgem has been investigated by flow cytometry analysis and protein expression profiling of L1210 wt cells treated in vitro with the squalenoyl gemcitabine bioconjugate. It was found that this nanomedicine arrested the cell cycle in G2/M, characterized by an increased cyclin A and cyclin E expression, and activation of caspase-3 and the cleavage of poly(ADP-ribose) polymerase with an increase of cytochrome C level. Taken together, these results suggest that the cell kill by this nanomedicine occurred through mitochondrial apoptotic triggered pathway, similarly to that of gemcitabine free.

Squalenoyl nanomedicine of gemcitabine is more potent after oral administration in leukemia-bearing rats: study of mechanisms

In an earlier report, we demonstrated the superior anticancer efficacy of orally administered squalenoyl gemcitabine (SQdFdC) nanomedicine over its parent drug gemcitabine on rats bearing RNK-16 large granular lymphocytic (LGL) leukemia. In the present communication, we investigated the mechanisms behind this observation both at the cell and tissue level. The mechanisms were investigated by performing cytotoxicity, cell uptake, and biodistribution experiments. In the presence of cytidine deaminase, SQdFdC nanoassemblies resisted deamination and exerted significant anticancer activity in vitro against RNK-16 LGL leukemia cells, whereas the cytotoxicity of free gemcitabine decreased by approximately 83-fold, indicating its degradation due to deamination. Additionally, the SQdFdC showed considerably higher intracellular accumulation and retention compared with gemcitabine (P<0.05). Unlike gemcitabine, the cellular access to SQdFdC was not influenced by nucleoside transporters. When administered orally to rats, unlike H-gemcitabine, the H-SQdFdC absorbed slowly, but exhibited an improved pharmacokinetics and tissue distribution profile, particularly in the lymphoid organs (the major organs of metastasis). The resistance to deamination, followed by the improved pharmacokinetic and tissue distribution, and greater accumulation and retention at the level of cancer cells, are the key factors for the superiority of SQdFdC nanoassemblies over free gemcitabine against RNK-16 LGL leukemia in rats.

Magnetoresponsive Squalenoyl Gemcitabine Composite Nanoparticles for Cancer Active Targeting

Gemcitabine is widely used against a variety of solid tumors; however, it possesses some important drawbacks such as rapid deamination leading to short biological half-life and induction of tumor resistance. We have shown previously that the covalent coupling of squalene (a precursor of cholesterol in sterol biosynthesis) to gemcitabine resulted in a potent nanomedicine, squalenoyl gemcitabine (SQdFdC), which displayed appreciable anticancer activity. Now, the present study describes the concept of magnetic responsiveness of SQdFdC nanoparticles obtained by the nanoprecipitation of SQdFdC around magnetite nanoparticles. To investigate these new core/shell nanoparticles, we have compared their structure, chemical composition and surface properties with those of either the magnetic core alone or of the SQdFdC coating material. X-ray diffraction and infrared spectroscopy studies have shown that the composite core/shell particles displayed an intermediate behavior between that of pure magnetite and of pure SQdFdC nanoparticles, whereas dark-field, high-resolution transmission electron microscopy allowed clear demonstration of the core/shell structure. Electrophoresis measurements as a function of both pH and ionic strength, as well as thermodynamic consideration, showed similar behavior of core/shell and pure SQdFdC nanoparticles, suggesting again the coating of the magnetite core by the SQdFdC prodrug. The two important parameters to be controlled in the efficient adsorption of SQdFdC onto magnetite nanocores were the magnetite/SQdFdC weight ratio and the pluronic F-68 concentration. Pluronic F-68 was found to play a key role as a surfactant in the generation of stable composite core/shell nanoparticle suspensions. Finally, the characterization of the magnetic properties of these core/shell nanoparticles revealed that if the squalenoyl shell reduced the magnetic responsiveness of the particles, it kept unchanged their soft ferrimagnetic character. Thus, the heterogeneous structure of these nanoparticles could confer them both magnetic field responsiveness and potential applicability as a drug carrier for active targeting to solid tumors.

Preclinical Toxicology (Subacute and Acute) and Efficacy of a New Squalenoyl Gemcitabine Anticancer Nanomedicine

This study investigates 1) the anticancer efficacy of a new squalenoyl prodrug of gemcitabine (SQgem) in nanoassembly form compared with gemcitabine at equitoxic doses and 2) the subacute and acute preclinical toxicity of these compounds. The toxicity studies revealed that SQgem nanoassemblies, like gemcitabine, were toxic, and they led to dose-dependent mortality after daily i.v. injections for 1 week, irrespective of the route of administration. However, a 4- to 5-day spaced dosing schedule (injections on day 0, 4, 8, and 13) was proved to be safer in terms of weight loss and hematological and other toxicity. Using this spaced dosing schedule, SQgem nanoassemblies exhibited impressive anticancer activity in mice bearing L1210 leukemia because this treatment led to 75% long-term survivors. In contrast, at equitoxic doses, neither free gemcitabine nor cytarabine led to longterm survivors and all the mice of these groups died of the disease. Further toxicity studies performed at lethal doses by blood and serum analysis and organ weight determinations revealed that the hematological toxicity was the dose-limiting toxicity in both SQgem nanoassemblies and gemcitabine, whereas probable gastrointestinal toxicity was also associated with free gemcitabine. The SQgem nanoassemblies did not display hepatotoxicity, which is one of the clinically encountered toxicities of gemcitabine. To summarize, these preclinical studies demonstrated that the toxicological profile of new squalenoyl gemcitabine nanomedicine was not distinct from that of the parent gemcitabine, whereas it was much more potent than gemcitabine at equitoxic doses and cytarabine at clinically relevant doses. These data support the candidature of SQgem for clinical trials.

Squalenoyl Nanomedicines as Potential Therapeutics

Nucleoside analogues display significant anticancer or antiviral activity by interfering with DNA synthesis. However, there are some serious restrictions to their use, including their rapid metabolism and the induction of resistance. We have discovered that the linkage of nucleoside analogues to squalene leads to amphiphilic molecules that self-organize in water as nanoassemblies of 100-300 nm, irrespective of the nucleoside analogue used. The squalenoyl gemcitabine exhibited superior anticancer activity in vitro in human cancer cells and gemcitabine-resistant murine leukemia cells, and in vivo in experimental leukemia both after intravenous and oral administration. The squalenoylation of other antiretroviral nucleosides also led to more potent drugs when tested in primary cultures of HIV-infected lymphocytes. Thus, the squalenoylation is an original technology platform for generating more potent anticancer and antiviral nanomedicines.