Sara Zalba

Pharmacodynamics of cisplatin-loaded PLGA nanoparticles administered to tumor-bearing mice

Biodegradable poly (lactic-co-glycolic) acid (PLGA) nanoparticles incorporating cisplatin have been developed to evaluate its in vivo efficacy in tumor-bearing mice. In vitro study proved two mechanisms of action for cisplatin depending on the dose and the rate at which this dose is delivered. In vivo study, 5mg/kg of cisplatin nanoparticles administered to mice, exhibited a tumor inhibition similar to free cisplatin, although the area under cisplatin concentration-time curve between 0 and 21days (AUC(0-21)) had lower value for the formulation than for drug solution (P<0.05). This result was associated with a higher activation of apoptosis in tumor, mediated by caspase-3, after nanoparticles administration. Toxicity measured as the change in body weight, and blood urea nitrogen (BUN) plasma levels showed that cisplatin nanoparticles treatment did not induce significant changes in both parameters compared to control, while for free drug, a statistical (P<0.01) increase was observed. In addition, a good correlation was found between time profiles of tumor volume and vascular endothelial growth factor (VEGF) plasma levels, suggesting that its expression could help to follow the efficacy of the treatment. Therefore, the PLGA nanoparticles seem to provide a promising carrier for cisplatin administration avoiding its side effects without a reduction of the efficacy, which was consistent with a higher activation of apoptosis than free drug.

Liposomes, a promising strategy for clinical application of platinum derivatives

Introduction: Liposomes represent a versatile system for drug delivery in various pathologies. Platinum derivatives have been demonstrated to have therapeutic efficacy against several solid tumors. But their use is limited due to their side effects. Since liposomal formulations are known to reduce the toxicity of some conventional chemotherapeutic drugs, the encapsulation of platinum derivatives in these systems may be useful in reducing toxicity and maintaining an adequate therapeutic response. Areas covered: This review describes the strategies applied to platinum derivatives in order to improve their therapeutic activity, while reducing the incidence of side effects. It also reviews the results found in the literature for the different platinum-drugs liposomal formulations and their current status. Expert opinion: The design of liposomes to achieve effectiveness in antitumor treatment is a goal for platinum derivatives. Liposomes can change the pharmacokinetic parameters of these encapsulated drugs, reducing their side effects. However, few liposomal formulations have demonstrated a significant advantage in therapeutic terms. Lipoplatin, a cisplatin formulation in Phase III, combines a reduction in the toxicity associated with an antitumor activity similar to the free drug. Thermosensitive or targeted liposomes for tumor therapy are also included in this review. Few articles about this strategy applied to platinum drugs can be found in the literature.

Application of different methods to formulate PEG-liposomes of oxaliplatin: evaluation in vitro and in vivo.

In this work, the Film Method (FM), Reverse-Phase Evaporation (REV), and the Heating Method (HM) were applied to prepare PEG-coated liposomes of oxaliplatin with natural neutral and cationic lipids, respectively. The formulations developed with the three methods, showed similar physicochemical characteristics, except in the loading of oxaliplatin, which was statistically lower (P<0.05) using the HM. The incorporation of a semi-synthetic lipid in the formulation developed by FM, provided liposomes with a particle size of 115 nm associated with the lowest polydispersity index and the highest drug loading, 35%, compared with the other two lipids, suggesting an increase in the membrane stability. That stability was also evaluated according to the presence of cholesterol, the impact of the temperature, and the application of different cryoprotectants during the lyophilization. The results indicated long-term stability of the developed formulation, because after its intravenous in vivo administration to HT-29 tumor bearing mice was able to induce an inhibition of tumor growth statistically higher (P<0.05) than the inhibition caused by the free drug. In conclusion, the FM was the simplest method in comparison with REV and HM to develop in vivo stable and efficient PEG-coated liposomes of oxaliplatin with a loading higher than those reported for REV.

Efficient gene delivery by EGF-lipoplexes in vitro and in vivo

AIMS In this work, we have evaluated the ability of targeted lipoplexes to enhance transgene expression in EGF receptor (EGFR) overexpressing tumor cells by using lipoplexes. MATERIALS & METHODS We prepared DOTAP/cholesterol liposomes modified with EGF at 0.5/1, 1/1, 2/1 and 5/1 lipid/DNA (+/-) charge ratio by sequentially mixing the liposomes with the ligand and adding the reporter or the therapeutic plasmid gene, pCMVLuc (pVR1216) or pCMVIL12, respectively. HepG2, DHDK12proB and SW620 cells were used for in vitro experiments, which were performed in the presence of 60% serum. RESULTS The characterization of EGF-lipoplexes indicated a size close to 300 nm and a variable net surface charge as a function of the amount of EGF associated to the cationic liposomes. EGF-lipoplexes, which showed an increased transfection activity, were positively charged, noncytotoxic and highly effective in protecting DNA from DNase I attack. Transfection activity in vitro resulted in an enhancement in the luciferase and IL-12 expression by EGF-lipoplexes compared with those without ligand (plain-lipoplexes) and to naked DNA. The results observed in SW620 cells, which are deficient in EGFR, confirmed that DNA uptake was predominantly via EGFR-mediated endocytosis. In vivo transfection activity was confirmed by luciferase imaging in living mice. Bioluminiscence could be detected mainly in the lung with a maximum signal 24 h after application. The resulting EGF-lipoplexes significantly increased the level of gene expression in mice compared with control or naked DNA. CONCLUSION These findings indicate that these nanovectors may be an adequate alternative to viral vectors for gene therapy.

Efficient serum-resistant lipopolyplexes targeted to the folate receptor

In this work, we have developed and evaluated a new targeted lipopolyplex (LPP), by combining polyethylenimine (PEI), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/Chol liposomes, the plasmids pCMVLuc/pCMVIL-12, and the ligand folic acid (FA), able to transfect HeLa and B16-F10 cells in the presence of very high concentration of serum (60% FBS). These complexes (Fol-LPP) have a net positive surface charge. The combination of folic acid with lipopolyplexes also enhanced significantly the transfection activity of the therapeutic gene interleukin-12 (IL-12), without any significant cytotoxicity. The specificity of the folate receptor (FR)-mediated gene transfer was corroborated by employing a folate receptor deficient cell line (HepG2). This formulation improved gene delivery showed by conventional lipoplexes or polyplexes resulting an efficient, simple, and nontoxic method for gene delivery of therapeutic genes in vitro and very probably in vivo.

Biopharmaceutic and pharmacodynamic modeling of the in vitro antiproliferative effect of new controlled delivery systems of cisplatin.

A biopharmaceutic-pharmacodynamic model is proposed to characterize the antiproliferative effect of controlled release formulations of cisplatin in cancer cell culture. In vitro release profiles from PLGA [poly(d,l-lactide-co-glycolide)] systems were described using a model based on the characterization of two drug release processes: diffusion and matrix degradation. Cytotoxicity data available consisting of the number of survival cells after a continuous exposure to free or encapsulated cisplatin were simultaneously modeled under the Gompertz framework incorporating the drug release model. The release model parameters showed that particle size was inversely related to the diffusion rate. The antiproliferative effect was described as a function of drug concentrations and exposure times. Two mechanisms were included: (i) an inhibition of cell proliferation, where cisplatin released from PLGA systems was mainly involved, followed by (ii) stimulation of cell death due to cisplatin activity and mediated by the activation of a signal transduction process. Cell accumulation in G2/M phase of the cell cycle followed by the activation of caspase-3, supported both mechanisms. The selected drug-effect model and its model parameters were independent from the formulation, which makes it a suitable tool to explore in silico, alternative in vitro and in vivo scenarios to optimize these delivery systems.

Immunoliposomes in clinical oncology: State of the art and future perspectives

&NA; Liposomal formulations entrapping a vast number of molecules have improved cancer therapies overcoming certain pharmacokinetic (PK) and pharmacodynamic limitations, many of which are associated with tumor characteristics. In this context, immunoliposomes represent a new strategy that has been widely investigated in preclinical cancer models with promising results, although few have reached the stage of clinical trials. This contrasts with the emerging clinical application of monoclonal antibodies (mAbs). This formulation allows the conjugation of different mAbs or antibody derivatives, such as monovalent variable fragments Fab’, to the polymers covering the surface of liposomes. The combination of this targeting strategy together with drug encapsulation in a single formulation may contribute to enhance the efficacy of these associated agents, reducing their toxicities. In this paper we will consider how factors such as particle size, lipid composition and charge, lipid‐polymer conjugation, method of production and type of ligand for liposome coupling influence the efficacy of these formulations. Furthermore, the high inter‐individual variability in the tumor microenvironment, as well as the poor experimental designs for the PK characterization of liposomes, make the establishment of the relationship between plasma or tumor concentrations and efficacy difficult. Thus, adequate dosing regimens and patient stratification regarding the target expression may contribute to enhance the possibility of incorporating these immunoliposomes into the therapeutic arsenal for cancer treatments. All these issues will be briefly dealt with here, together with a section showing the state of the art of those targeted liposomes that are coming up for testing in clinical trials. Finally, some insights into future developments such as the combination of specificity and controlled release, based on the application of different stimuli, for the manipulation of stability and cargo release, will be offered. This has been included in order to highlight the new opportunities for targeted liposomes, including immunoliposomes. Graphical abstract Figure. No Caption available. Abbreviations: ABC: Accelerated blood clearance; BBB: Blood brain barrier; CH: Cholesterol; CHEMS: Cholesteryl hemisuccinate; DOPE: 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine; DPPC: 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine; DPPG2: 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphodiglycerol; DSPC: 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine; DSPE‐PEG: 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[amino(polyethylene glycol)‐2000; EGFR: Epidermal growth factor receptor; EPH: Erythropoietin‐producing hepatoma; Fab′: Monovalent fragment antigen binding; (Fab′)2: Bivalent fragment antigen binding; Fc: Fragment crystallizable; HER‐2: Human epidermal growth factor receptor‐2; HIFU: High intensity focused ultrasound; HSPC: l‐&agr;‐phosphatidylcholine hydrogenated (Soy); Hz‐PEG: Hydrazide‐polyethylenglycol; Lyso‐PC: Lysophosphatidylcholine; Mal‐PEG: Maleimide‐polyethylenglycol; MEA: 2‐Mercaptoethylamine·HCl; MMP‐1: Matrix metalloproteinase‐1; MMP‐8: Matrix metalloproteinase‐8; NGPE: N‐glutaryl‐phosphatidylethanolamine; PDP‐PEG: Pyridylditiopropionoylamino polyethylenglycol; PEG: Polyethylene glycol; RES: Reticuloendotelial system; ROS: Reactive oxygen species; scFv: Single chain variable fragment; sdAbs: Single domain antibodies; siRNA: Small interfering RNA; TGF&bgr;: Transforming growth factor &bgr;; Tm: Melting temperature; TNF&agr;: Tumor necrosis factor &agr;; TSL: Thermosensitive liposome; VEGF: Vascular endothelial growth factor.

EGF-liposomes promote efficient EGFR targeting in xenograft colocarcinoma model.

AIM Development of EGF-liposomes (LP-EGF) for selective molecules delivery in tumors expressing EGFR. MATERIAL & METHODS In vitro cellular interaction of EGF-LP and nontargeted liposomes (LP-N) was assayed at 37 and 4 °C in cells expressing different EGFR levels. Receptor-mediated uptake was investigated by competition with a monoclonal antibody anti-EGFR. Selective intracellular drug delivery and efficacy was tested by oxaliplatin encapsulation. In vivo biodistribution of LP-N and LP-EGF was done in xenograft model. RESULTS LP-EGF was internalized by an active and selective mechanism through EGFR without receptor activation. Oxaliplatin LP-EGF decreased IC50 between 48 and 13% in cell EGFR+. LP-EGF was accumulated in tumor over 72 h postdosing, while LP-N in spleen. CONCLUSION LP-EGF represents an attractive nanosystem for cancer therapy or diagnosis.

Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody.

Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting.