[Digital transformation of perioperative nurse-coordinated protocols in renal surgery for enhanced recovery and outpatient surgery using UroConnect® application]. / Digitalisation de la coordination infirmière des parcours périopératoires de chirurgie rénale en réhabilitation améliorée et ambulatoire par l'application UroConnect®.

Robot Assisted Partial Nephrectomy (RAPN) is a standard of care for localized renal tumors. It allows a good carcinological control while limiting complications. Despite numerous benefits, the economic sustainability of robotic assistance remains a challenge in the French health care system. The introduction in our institution of two perioperative nurse-coordinated protocols for patients undergoing RAPN (Enhanced Recovery After Surgery: NP-RAAC in 2015 and Outpatient: Ambu-Rein in 2016) is associated with a shortening of the average length of hospital stay, thus reducing the cost of robotic assisted procedures. With the aim of improving efficiency of nursing support within these protocols, we have introduced digitalized nursing coordination by developing a urological perioperative application: UroConnect®. This device is offered to patients by the coordinating nurses during a preoperative visit. It provides information on the pathology and its surgical management. Self-completed questionnaires sent at key moments collect data from the first month after surgery and detect patients presenting difficulties or complications, allowing the nurses to respond with appropriate care. The application allows a secure discharge, a personalised follow-up and an increase in the patient's autonomy and compliance with care.

pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in quasi-anhydrous samples.

pH-sensitive liposomes made of dioleoylphosphatidylethanolamine (DOPE)/oleic acid (OA)/cholesterol (CHOL) mixtures were shown to be very promising carriers for oligonucleotides (ON). However, it appeared necessary to clarify the structural consequence of the interactions of ON with the liposome, and especially on DOPE, the lipid responsible for the pH sensitivity. The present study was carried out by differential scanning calorimetry and X-ray diffraction, at low hydration. In such a case, DOPE generally adopt a hexagonal phase. It could be shown that ON increased DOPE transition temperature and increased v/al, as a result of electrostatic interactions between ON and DOPE headgroups. OA was found to have exactly opposite effects, its presence between DOPE molecules inhibiting the formation of hydrogen bonds. The presence of both ON and OA allowed the system to organize in a lamellar phase below the solid/liquid transition, whereas above this temperature ON preferably interacted with DOPE in a hexagonal phase and led OA to separate.

Reversion of multidrug resistance by co-encapsulation of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles.

Individual and combined polyalkylcyanoacrylate nanoparticle formulation of cyclosporin A and doxorubicin were prepared and evaluated in an attempt to show improved growth inhibition efficacy in a resistant cell culture line. The drug loaded nanoparticles were prepared using the well established emulsion polymerization process without using any modification for the hydrophilic doxorubicin drug whereas the incorporation of cyclosporin A needed to wait a moment after the polymerization reaction started. This was necessary to avoid cyclosporin A precipitation and polymer aggregation. Cyclosporin A release from the nanoparticles was rapid probably because the drug was adsorbed onto the nanoparticles surface rather than embedded into the polymeric core. Doxorubicin displayed also a burst effect but with a slower second phase probably related with the nanoparticles bioerosion rate owing to its entrapment in the polymeric network. Finally, it was shown in resistant cell culture experiments that the association of both cyclosporin A and doxorubicin within a single nanoparticle formulation elicited the most effective growth rate inhibition as compared to other combinations of both drugs while using a lower amount of polymer compared to separated nanoparticle formulations. This result was probably due to the synergistic effect achieved by combining the chemo-sensitizing compound cyclosporin A, with an effective cytotoxic drug like doxorubicin.

On the mechanism of action of doxorubicin encapsulation in nanospheres for the reversal of multidrug resistance.

We had previously shown that doxorubicin encapsulation in polyisohexylcyanocrylate nanospheres could circumvent the P-glycoprotein-mediated multidrug resistance (MDR) exhibited by C6 rat glioblastoma in culture. We then investigated what could be the mechanism of such a circumvention. The cytotoxicity of free and encapsulated doxorubicin was evaluated in two MDR variants of the C6 cell line in a device allowing the separation of cells from drugs by a polycarbonate membrane of 0.2 micron pore size. We observed that the progressive disruption of the nanospheres allowed their doxorubicin content to reach the cell monolayer and exert its cytotoxicity in a fashion similar to that exhibited by free doxorubicin. However, no circumvention of MDR is obtained by doxorubicin encapsulation when drug-containing nanospheres are separated from the cells by the polycarbonate membrane. In addition, no effect on azidopine binding to P-glycoprotein-enriched membranes is exerted by unloaded nanospheres, even after their spontaneous degradation in cell-culture medium. Taken together, these results suggest that a physical contact between doxorubicin-containing nanospheres and the cells is required for the circumvention of MDR. The role of degradation products from the nanospheres as modulators of P-glycoprotein activity can be ruled out.

Uptake of doxorubicin from loaded nanoparticles in multidrug-resistant leukemic murine cells.

Previous studies have clearly demonstrated that polyisobutylcyanoacrylate (PIBCA) doxorubicin-loaded nanoparticles (NS-Dox PIBCA) can overcome the resistance of P388/ADR cells. In the present paper, we found that overcoming multidrug resistance with the aid of doxorubicin (Dox) loaded onto these nanoparticles was associated with an increased intracellular drug content. Indeed, after a 6-h incubation period, the amount of cell-associated drug was 5 times higher when the cells were incubated with NS-Dox PIBCA as compared with free Dox. Further experiments, such as uptake studies in the presence of cytochalasin B or efflux studies, indicated a possible mechanism of nanoparticle/cell interaction. These results suggested that nanoparticles did not enter the cells by an endocytic process, in contrast to a previous hypothesis.

Investigation of the role of macrophages on the cytotoxicity of doxorubicin and doxorubicin-loaded nanoparticles on M5076 cells in vitro.

Doxorubicin-loaded PACA nanoparticles have been shown to be more efficient than free drug in mice bearing hepatic metastasis of the M5076 tumour. Due to the high phagocytic activity of Küpffer cells in the liver, it may be that these cells played a role of drug reservoir after nanoparticle phagocytosis. Therefore, the objective of this study was to assess the role of macrophages in mediating the cytotoxicity of doxorubicin-loaded nanoparticles on M5076 cells. The growth inhibition of tumour cells was evaluated in two ways: firstly, the cells were incubated in a coculture system consisting of special wells with two compartments separated by a porous membrane. M5076 cells were seeded into the lower compartment and the macrophages J774.A1 were introduced into the upper part. The macrophages were activated or not by IFN-gamma. The drug preparations were added only in the macrophage insert. Secondly, growth inhibition was also assessed in the conventional way, i.e. in direct contact with the tumour cells to serve as a reference. After direct contact, free doxorubicin (Dox) and doxorubicin-loaded nanoparticles (NP-Dox) had the same efficacy against M5076 cell growth. The coculture experiments led to a 5-fold increase in the IC(50) for both Dox and NP-Dox. The activation of macrophages by IFN-gamma in coculture significantly decreased the IC(50) values. In conclusion, after phagocytosis of doxorubicin-loaded nanoparticles, J774.A1 cells were able to release active drug, allowing it to exert its cytotoxicity against M5076 cells. Drug efficacy was potentiated by the activation of macrophages releasing cytotoxic factors such as NO, which resulted in increased tumour cell death. Thereby, the coculture system permitted us to investigate the macrophage-mediated cytotoxicity of colloidal carriers loaded with an anticancer drug, which is of great interest when further i.v. administration is envisaged.

Drug delivery to resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles.

Simultaneous cellular resistance to multiple lipophilic drugs represents a major problem in cancer chemotherapy. This drug resistance may appear clinically either as a lack of tumor size reduction or as the occurrence of clinical relapse after an initial positive response to antitumor treatment. The resistance mechanism can have different origins either directly linked to specific mechanisms developed by the tumor tissue or connected to the more general problem of distribution of a drug towards its targeted tissue. The purpose of this paper is to summarize the results of the use of poly(alkyl cyanoacrylate) nanoparticles to overcome multidrug resistance (MDR) phenomena at both the cellular and the non-cellular level.

Cationic emulsions improves the delivery of oligonucleotides to leukemic P388/ADR cells in ascite.

The aim of this study was to investigate the in vivo ability of O/W cationic emulsions to deliver oligonucleotides (ON) in leukemic P388/ADR cells in ascite, after intraperitoneal (IP) administration in mice. Cationic emulsions were prepared by microfluidization as previously described by Teixeira et al. [Pharm. Res 16 (1999) 30]. The formulations consisted mainly of medium chain triglycerides, phosphatidylcholine (PC), poloxamer, and either a monocationic lipid stearylamine (PC/SA-emulsion) or a polycationic lipid RPRC(18) (PC/RPRC(18)-emulsion). A model ON (33P-pdT(16)) was associated with cationic emulsions by single addition at the end of the manufacturing process. Seven days after P388/ADR inoculation IP to mice, ON free or associated with PC/SA or PC/RPRC(18) emulsions was injected IP at a dose of 0.5 mg/kg. At different interval times, ascite including cells, blood and the main organs were collected and the radioactivity counted by liquid scintillation. The overall results showed significantly high amounts of ON in the leukemic cell pellet, 24 h after administration of ON associated to either PC/SA (AUC(0-24 h)=13634, %injected dose/min) or PC/RPRC(18) (AUC(0-24 h)=22592, % injected dose/min), contrary to the free ON solution (AUC(0-24 h)=3095, %injected dose/min), which displayed only reduced capture by cancer cells. In conclusion, complexation of ON with cationic emulsions had a beneficial effect in increasing tumor cells uptake in vivo (up to sevenfold for PC/RPRC(18)-emulsion) after IP administration. This could open interesting prospects for the treatment of ovarian cancers.

Factors influencing the oligonucleotides release from O-W submicron cationic emulsions.

We recently described a positively charged O-W emulsion as a delivery system for oligonucleotides (ON) [Teixeira et al., Pharm. Res. 16 (1999) 30-36]. The present paper investigates the role of the main formulation parameters that may have an influence on the release-rate of a model ON in a protein-containing medium, i.e. the nature of the oily core, the presence of pegylated lipids, the lipid phase transition temperature, and the cationic lipid structure. The use of cationic lipids bearing diacyl chains (and especially polycations) appeared as the only efficient strategy to reduce the ON release rate. In order to have a better insight on the nature of the interactions between the ON and the interfacial lipids, adsorption isotherms at the air-water interface, fluorescence resonance energy transfer and zeta-potential measurements have been performed. Electrostatic interactions were found to play a crucial role. In contrast, the incorporation of PEG-phospholipids acted as a barrier and maintained the ON molecules distant from the interface, leading to a more rapid release. Finally, ON integrity was assessed by a competitive hybridization assay. The results suggest the existence of a transient ion-pair (ON-cationic lipids) protecting ON against nuclease degradation even after its release from the emulsions.

pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in excess water.

The cytoplasmic delivery of drugs encapsulated into pH-sensitive liposomes is under the control of a lamellar-to-hexagonal transition. In a previous study, under anhydrous conditions, oligonucleotides (ODN) encapsulated in pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine (DOPE)/oleic acid (OA)/cholesterol (CHOL) were shown to modify the phase behaviour of DOPE. In the present study, the lipid/ODN interactions were evaluated in fully hydrated samples by surface tension measurements, differential scanning calorimetry, X-ray diffraction and turbidimetry. Concerning the lipids, it was shown that OA provoked a disorganisation of DOPE lamellar phases and led to the complete disappearance of hexagonal transition along with heating. The addition of CHOL further decreased the lipid packing in the bilayers. Concerning ODN, these molecules provoked an increase in the surface pressure of a DOPE/OA/CHOL monolayer, indicating the existence of molecular interactions with the lipids. At a supramolecular level, ODN induced a more ordered organisation of DOPE molecules in the lamellar and hexagonal phases, and completely abolished the disorganisational effect of OA and CHOL.

Characterization of oligonucleotide/lipid interactions in submicron cationic emulsions: influence of the cationic lipid structure and the presence of PEG-lipids.

We have recently described how oligonucleotide (ON) stability and release from O/W cationic emulsions are governed by the lipid composition. The aim of the present paper was to investigate the properties of the ON/lipid complexes through fluorescence resonance energy transfer (FRET), size, surface tension measurements and cryomicroscopy. Starting from a typical emulsion containing stearylamine as a cationic lipid, the influence of the lipid structure (monocationic molecules bearing mono or diacyl chains, or polycations) as well as of the presence of PEGylated lipids, were studied. The presence of a positive charge on the droplet surface clearly contributed to enhance the ON interaction with lipid monolayers and to bring the ON molecules closer to the interface. Hydrophobic interactions through the acyl chains were shown to further enhance the anchorage of the ON/lipid complexes. In contrast, the incorporation of PEGylated lipids acted as a barrier against the establishment of electrostatic bindings, the polyethyleneglycol chains acting themselves as interaction sites for the ON leading to hydrophilic complexes. Similar features were observed for the polycationic lipid, and cryomicroscopy revealed the existence of bridges of various intensities between the droplets of the emulsion containing either PEG or the polycation, probably because of the configuration of the ON at the interface.

Ibuprofen-loaded ethylcellulose microspheres: analysis of the matrix structure by thermal analysis.

The determination of the drug dispersion state in microspheres prepared by the solvent evaporation method is essential to foresee the stability of the particles and the drug release behavior. The present work deals with ibuprofen-loaded ethylcellulose microspheres, that are characterized by a lower drug melting point than the polymer glass transition temperature. Although annealing experiments were not possible, the study has evidenced the presence of a metastable molecular dispersion for intermediate loadings, coexisting with a solid solution and a crystalline dispersion of the drug in the polymer matrix. In addition, differential scanning calorimetry helped to distinguish between surface and inner ibuprofen crystals, which interact differently with the polymer matrix and therefore have different melting points.

Pulmonary administration of IgG loaded liposomes for passive immunoprophylaxy.

Local passive immunoprophylaxy has been used in pulmonary infectious diseases successfully. However, the short immunoglobulins half-life in the lungs limits the duration of their action. The aim of the present study was to evaluate the efficiency of human polyvalent intravenous immunoglobulins (IVIG) when protected after encapsulation within EPC: DPPG liposomes by dehydration/rehydration. Two IVIG concentrations were chosen: 10 and 1 mg/ml for further studies in mice infected by influenza A. For the highest concentration (10 mg/ml), IVIG loaded liposomes did not significantly differ from IVIG/unloaded liposomes mixture with around 45% association yield. For the lowest concentration (1 mg/ml), two thirds of the IVIG associated were found inside the vesicles. In vivo, IVIG administered intranasally at 10 mg/ml (500 microg per mouse) 4 days before the infection led to 100% survival whatever the formulation. When administered at a lower dose (1 mg/ml-50 microg per mouse) 2 days before the challenge, loaded liposomes were found less efficient than free IVIG while unloaded liposomes showed a slight aspecific immunoprotection. Gastrointestinal clearance must be responsible for a major loss of liposomes compared to IVIG solution because of a higher viscosity of the formulation. Discrepancies with the literature are discussed.

Prodrug-based intracellular delivery of anticancer agents.

There are numerous anticancer agents based on a prodrug approach. However, no attempt has been made to review the ample available literature with a specific focus on the altered cell uptake pathways enabled by the conjugation and on the intracellular drug-release mechanisms. This article focuses on the cellular interactions of a broad selection of parenterally administered anticancer prodrugs based on synthetic polymers, proteins or lipids. The report also aims to highlight the prodrug design issues, which are key points to obtain an efficient intracellular drug delivery. The chemical basis of these molecular concepts is put into perspective with the uptake and intracellular activation mechanisms, the in vitro and in vivo proofs of concepts and the clinical results. Several active targeting strategies and stimuli-responsive architectures are discussed throughout the article.

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.

From nanotechnology to nanomedicine: applications to cancer research.

Scientific advances have significantly improved the practice of medicine by providing objective and quantitative means for exploring the human body and disease states. These innovative technologies have already profoundly improved disease detection, imaging, treatment and patient follow-up. Today's analytical limits are at the nanoscale level (one-billionth of a meter) enabling a detailed exploration at the level of DNA, RNA, proteins and metabolites which are in fact nano-objects. This translational review aims at integrating some recent advances from micro- and nano-technologies with high potential for improving daily oncology practice.

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.

Freeze-drying of squalenoylated nucleoside analogue nanoparticles.

Nucleoside analogues are potent anticancer or antiviral agents that however display some limitations (rapid metabolism, induction of resistance). In order to overcome these drawbacks, we recently proposed new prodrugs, in which nucleoside analogues were covalently coupled to squalene (SQ). The resulting amphiphilic compounds spontaneously formed nanoparticles (NPs) and displayed a promising efficacy both in vitro and in vivo. Since long-term stability is essential for further clinical development we needed to develop a laboratory-scale freeze-drying protocol in order to improve the colloidal stability of those NPs. Squalenoylated gemcitabine (SQdFdC) has been successfully freeze-dried with trehalose (10%, w/w) as a cryoprotectant. Concentrations of SQdFdC up to 4mg/mL after freeze-drying and rehydration have been obtained, which is necessary for in vivo studies. Stability measurements by dynamic light scattering showed that trehalose had a stabilizing effect on SQdFdC NPs, and that freeze-dried SQdFdC NPs could be stored up to four months at room temperature before rehydration, without loss of stability. In vitro cytotoxicity studies on three murine cell lines showed that SQdFdC NPs retained their cytotoxic activity after freeze-drying. We showed that this freeze-drying protocol could also be applied to squalenoylated didanosine (SQddI) and zalcitabine (SQddC). Overall, these results allow for the use of freeze-dried NPs in upcoming preclinical trials of the different squalenoylated compounds developed in our laboratory.

Ibuprofen-loaded ethylcellulose microspheres: release studies and analysis of the matrix structure through the Higuchi model.

The following work deals with ibuprofen-loaded ethylcellulose microspheres. The drug exists either in a state of molecular dispersion or in crystalline form, depending on the encapsulation ratio. The in vitro release profiles have been studied and the Higuchi model applied to the experimental results. With an appropriate treatment of the results, it has been shown that the surface crystals responsible for the observed burst effect are really encapsulated by the polymer. The calculation of the tortuosity factor clearly shows that the release kinetics are controlled by the hydrophobicity of ethylcellulose and the geometry of the porous volume resulting from the dissolution of ibuprofen crystals. It thus appears that crystals are probably differently distributed in the matrix depending on the microsphere size.

Oligonucleotides encapsulated in pH sensitive liposomes are efficient toward Friend retrovirus.

Retroviruses present multiple RNA targets for antisense oligonucleotides. An oligodesoxyribonucleotide (15 mer) complementary to the region of the initiation codon AUG of the env gene mRNA of Friend retrovirus was an inhibitor of the translation of Env protein in vitro. No effect was observed on cells infected with Friend retrovirus. We observed that these oligomers were rapidly degraded in cellular medium. After encapsulation into liposomes, they inhibited the spreading of the virus for chronic or de novo infection. We have compared the efficiency of two compositions of liposomes: pH sensitive and non pH sensitive formulations. Oligomers encapsulated in pH sensitive liposomes were more active that those encapsulated in non pH sensitive liposomes. pH sensitive liposomes could allow to avoid degradation of oligomers by lysosomes.