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The aggressiveness of melanoma and lack of effective therapies incite the discovery of novel strategies. Recently, a new dual acting hybrid molecule (HM), combining a triazene and a ʟ-tyrosine analogue, was synthesized. HM was designed to specifically be activated by tyrosinase, the enzyme involved in melanin biosynthesis and overexpressed in melanoma. HM displayed remarkable superior antiproliferative activity towards various cancer cell lines compared with temozolomide (TMZ), a triazene drug in clinical use, that acts through DNA alkylation. In B16-F10 cells, HM induced a cell cycle arrest at phase G0/G1 with a 2.8-fold decrease in cell proliferation index. Also, compared to control cells, HM led to a concentration-dependent reduction in tyrosinase activity and increase in caspase 3/7 activity. To maximize the therapeutic performance of HM in vivo, its incorporation in long blood circulating liposomes, containing poly(ethylene glycol) (PEG) at their surface, was performed for passively targeting tumour sites. HM liposomes (LIP HM) exhibited high stability in biological fluids. Preclinical studies demonstrated its safety for systemic administration and in a subcutaneous murine melanoma model, significantly reduced tumour progression. In a metastatic murine melanoma model, a superior antitumour effect was also observed for mice receiving LIP HM, with markedly reduction of lung metastases compared to positive control group (TMZ). Biodistribution studies using 111 In-labelled LIP HM demonstrated its ability for passively targeting tumour sites, thus correlating with the high therapeutic effect observed in the two experimental murine melanoma models. Overall, our proposed nanotherapeutic strategy was validated as an effective and safe alternative against melanoma., Competing Interests: Conflict of interest statement The authors report there are no competing interests to declare., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Strategies targeting nucleolin have enabled a significant improvement in intracellular bioavailability of their encapsulated payloads. In this respect, assessment of the impact of target cell heterogeneity and nucleolin homology across species (structurally and functionally) is of major importance. This work also aimed at mathematically modelling the nucleolin expression levels at the cell membrane, binding and internalization of pH-sensitive pegylated liposomes encapsulating doxorubicin and functionalized with the nucleolin-binding F3 peptide (PEGASEMP), and resulting cytotoxicity against cancer cells from mouse, rat, canine, and human origin. Herein, it was shown that nucleolin expression levels were not a limitation on the continuous internalization of F3 peptide-targeted liposomes, despite the saturable nature of the binding mechanism. Modeling enabled the prediction of nucleolin-mediated total doxorubicin exposure provided by the experimental settings of the assessment of PEGASEMP's impact on cell death. The former increased proportionally with nucleolin-binding sites, a measure relevant for patient stratification. This pattern of variation was observed for the resulting cell death in nonsaturating conditions, depending on the cancer cell sensitivity to doxorubicin. This approach differs from standard determination of cytotoxic concentrations, which normally report values of incubation doses rather than the actual intracellular bioactive drug exposure. Importantly, in the context of development of nucleolin-based targeted drug delivery, the structural nucleolin homology (higher than 84%) and functional similarity across species presented herein, emphasized the potential to use toxicological data and other metrics from lower species to infer the dose for a first-in-human trial., (© 2021. Controlled Release Society.)

Stem cells have received considerable attention by the scientific community because of their potential for tissue engineering and regenerative medicine. The most frequently used method to promote their differentiation is supplementation of the in vitro culture medium with growth/differentiation factors (GDFs). The limitations of that strategy caused by the short half-life of GDFs limit its efficacy in vivo and consequently its clinical use. Thus, the development of new concepts that enable the bioactivity and bioavailability of GDFs to be protected, both in vitro and in vivo, is very relevant. Nanoparticle-based drug delivery systems can be injected, protect the GDFs and enable spatiotemporal release kinetics to be controlled. Liposomes are well-established nanodelivery devices presenting significant advantages, viz. a high load-carrying capacity, relative safety and easy production, and a versatile nature in terms of possible formulations and surface functionalization. The main objective of the present study was to optimize the formulation of liposomes to encapsulate dexamethasone (Dex). Our results showed that the optimized Dex-loaded liposomes do not have any cytotoxic effect on human bone marrow-derived mesenchymal stem cells (hBMSCs). More importantly, they were able to promote an earlier induction of differentiation of hBMSCs into the osteogenic lineage, as demonstrated by the expression of osteoblastic markers, both phenotypically and genotypically. We concluded that Dex-loaded liposomes represent a viable nanoparticle strategy with enhanced safety and efficacy for tissue engineering and regenerative medicine., (Copyright © 2013 John Wiley & Sons, Ltd.)

Mitoxantrone-based combinations are a standard palliative treatment in hormone-refractory prostate cancer (HRPC) but with no survival benefit. Imatinib has shown preclinical activity against HRPC although minimal clinical therapeutic efficacy. Our previous in vitro studies demonstrated that simultaneous combination of imatinib with mitoxantrone yielded additive growth inhibition effects against PC-3 cell line. The main aim of the work was to develop novel liposomal formulations comprising imatinib co-encapsulated with mitoxantrone, by different loading methods and experimental conditions, in order to achieve the highest drug loading and maximum physical stability. In the optimized formulations, imatinib and mitoxantrone were actively co-loaded by means of a (NH4)2SO4 transmembrane gradient. Encapsulation efficiency, mean size diameter and drug retention in storage and in biological conditions were characterized. Our study presented for the first time an active loading method for imatinib and suggests that the optimized liposomal formulation co-encapsulates both drugs with high encapsulation efficiency (> 95%), shows enhanced drug retention under tested conditions and delivers a drug:drug ratio capable of improving tumor cell growth inhibition with a mitoxantrone dose reduction of 2.6-fold as compared to single liposomal formulation. Therefore, our nanotechnology-based drug combined platform may constitute a promising strategy in prostate cancer therapy.

A new approach to enzyme-responsive MRI agents based on the use of liposomes loaded with a high number of paramagnetic metal complexes (Gd-HPDO3A) is presented. It relies on the disruption of low relaxivity aggregates formed by liposomes and a macromolecular substrate that is selectively cleaved by the enzyme of interest. The interaction of anionic liposomes composed of POPC:CHOL:DPGS and the cationic protein protamine yields a poorly soluble supramolecular assembly endowed with a low relaxivity. The action of the serine protease trypsin causes the digestion of protamine and the consequent de-assembly of the supramolecular adduct. The process is accompanied by an overall relaxation enhancement of solvent water protons as consequence of the dissolution of the aggregated liposomes. The observed increase of relaxivity is linearly dependent on the enzyme concentration. An illustrative example of the possible use of the herein presented responsive agent has been reported. It consists of the entrapment of the supramolecular assembly in alginate microcapsules that have often been used as envelopes for in vivo applications of stem cells and pancreatic islets. The change in the observed longitudinal relaxation rate R(1) (leading to an hyperintense signal in the corresponding MR images) may act as a sensor of the protease activity in the biological environment in which the capsules is located., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Chronic myeloid leukemia (CML) is triggered by the BCR-ABL oncogene. Imatinib is the first-line treatment of CML; however imatinib resistance and intolerance have been detected in many patients. Therefore, new therapeutic approaches are required. The present work aimed at the development and application of transferrin receptor (TrfR) targeted liposomes co-encapsulating anti-BCR-ABL siRNA and imatinib at different molar ratios. The encapsulation yields and drug loading of each molecule was evaluated. Anti-leukemia activity of the developed formulations co-encapsulating siRNA and imatinib and of the combination of Trf-liposomes carrying siRNA and free imatinib under two different treatment schedules of pre-sensitization was assessed. The results obtained demonstrate that the presence of imatinib significantly decreases the encapsulation yields of siRNA, whereas imatinib encapsulation yields are increased by the presence of siRNA. Cytotoxicity assays demonstrate that the formulations co-encapsulating siRNA and imatinib promote a 3.84-fold reduction on the imatinib IC(50) (from 3.49 to 0.91 µM), whereas a 8.71-fold reduction was observed for the pre-sensitization protocols (from 42.7 to 4.9 nM). It was also observed that the formulations with higher siRNA to imatinib molar ratios promote higher cell toxicity. Thus, the present work describes a novel triple targeting strategy with one single system: cellular targeting (through the targeting ligand, transferrin) and molecular targeting at the BCR-ABL mRNA and Bcr-Abl protein level., (© 2010 Wiley Periodicals, Inc.)

Anticancer systemic gene silencing therapy has been so far limited by the inexistence of adequate carrier systems that ultimately provide an efficient intracellular delivery into target tumor cells. In this respect, one promising strategy involves the covalent attachment of internalizing-targeting ligands at the extremity of PEG chains grafted onto liposomes. Therefore, the present work aims at designing targeted liposomes containing nucleic acids, with small size, high encapsulation efficiency and able to be actively internalized by SCLC cells, using a hexapeptide (antagonist G) as a targeting ligand. For this purpose, the effect of the liposomal preparation method, loading material (ODN versus siRNA) and peptide-coupling procedure (direct coupling versus post-insertion) on each of the above-mentioned parameters was assessed. Post-insertion of DSPE-PEG-antagonist G conjugates into preformed liposomes herein named as stabilized lipid particles, resulted in targeted vesicles with a mean size of about 130 nm, encapsulation efficiency close to 100%, and a loading capacity of approximately 5 nmol siRNA/mumol of total lipid. In addition, the developed targeted vesicles showed increased internalization in SCLC cells, as well as in other tumor cells and HMEC-1 microvascular endothelial cells. The improved cellular association, however, did not correlate with enhanced downregulation of the target protein (Bcl-2) in SCLC cells. These results indicate that additional improvements need to be performed in the future, namely by ameliorating the access of the nucleic acids to the cytoplasm of the tumor cells following receptor-mediated endocytosis., (Copyright 2009 Elsevier B.V. All rights reserved.)

RNA interference provides a powerful technology for silencing any single protein within a target cell. Therapeutic applications of small interfering RNAs (siRNAs), however, require vehicles for stable and efficient delivery of these nucleic acid molecules, both in vitro and in vivo. Targeted lipoplexes have been used as a promising system to mediate siRNA delivery and to achieve gene silencing. Electrostatic association of transferrin (Tf) to cationic liposomes enhances the transfection of siRNA. We describe the methods used for the preparation of Tf-lipoplexes and evaluation of their biocompatibility. Approaches to assess the complexation of siRNAs, the ability of Tf-lipoplexes to mediate siRNA protection, and intracellular delivery, as well as to achieve both mRNA and protein knockdown, are also described. We illustrate the efficiency of Tf-lipoplexes in mediating the knockdown of both green fluorescent protein (GFP) and luciferase in cell lines stably expressing these reporter genes.

The main aim of this work was to develop novel targeted sterically stabilised pH-sensitive liposomes tailored to promote efficient intracellular delivery of therapeutic molecules into human T-leukaemia cells. Our results indicate that the targeting moiety (thiolated transferrin) was successfully coupled to the distal reactive maleimide terminus of poly(ethylene glycol)-phospholipid conjugates incorporated in the liposomal bilayer. Results from atomic force microscopy studies, performed to characterise vesicle surface topology, indicated that, to a certain extent, thiolated transferrin has the ability to associate in a non-specific manner with the lipid membrane of pegylated liposomes. This is an issue not commonly reported in the literature but which is crucial to demonstrate the targeting proof of principle. Nevertheless, fluorimetric studies together with confocal microscopy clearly demonstrate that liposomes bearing covalently coupled transferrin associate more extensively to human T-leukaemia cells in vitro than non-targeted liposomes. Cell mechanistic studies indicate that targeted liposomes bind specifically to transferrin receptors and are internalised via receptor-dependent endocytotic pathway. In addition, the biophysical features exhibited by the developed liposomes, namely their ability to promote pH-triggered cytoplasmic delivery of loaded material, make them promising delivery systems for in vivo targeting of therapeutic molecules to tumours.

Strategies used to enhance liposome-mediated drug delivery in vivo include the enhancement of stability and circulation time in the bloodstream, targeting to specific tissues or cells, and facilitation of intracytoplasmic delivery. pH-sensitive liposomes have been developed to mediate the introduction of highly hydrophilic molecules or macromolecules into the cytoplasm. These liposomes destabilize under acidic conditions found in the endocytotic pathway, and usually contain phosphatidylethanolamine (PE) and titratable stabilizing amphiphiles. Formulations without PE have also been developed. Encapsulated compounds are thought to be transported into the cytoplasm through destabilization of or fusion with the endosome membrane. Incorporation of a low mole percentage of poly(ethylene glycol) (PEG)-conjugated lipids into pH-sensitive liposomes confers prolonged circulation times to these liposomes, which are otherwise cleared rapidly. While the incorporation of PEG-lipids reduces the pH-dependent release of encapsulated fluorescent markers in vitro, it does not hinder the cytoplasmic delivery of the markers per cell-associated liposome. This suggests that intracellular delivery is not dictated simply by the destabilization of the liposomes. Antibodies or ligands to cell surface receptors can be coupled to pH-sensitive or sterically stabilized pH-sensitive liposomes for targeting. pH-sensitive liposomes have been used to deliver anticancer drugs, antibiotics, antisense oligonucleotides, ribozymes, plasmids, proteins and peptides to cells in culture or in vivo.

NeoPharm is developing liposome-encapsulated, c-Raf antisense oligodeoxynucleotides (LErafAON) for the potential treatment of various solid tumors, including those that have become resistant to radiation or chemotherapy. Phase I/II trials commenced in March 2001 and were ongoing as of June 2003.

Purpose: Simple methods for the large-scale manufacture of ligand-targeted liposomes will be needed if clinical trials are to proceed. We tested a recently developed technology for inserting peptide ligands into preformed Stealth liposomes. Antagonist G-targeted liposomes (PLG) were prepared and loaded with doxorubicin and their cellular association and cytotoxicity were evaluated using the human small cell lung cancer H69 cell line., Methods: The hexapeptide antagonist G was covalently coupled via a thioether bond to the terminus of polyethylene glycol (PEG) in micelles formed from maleimide-derivatized poly(ethylene glycol) (Mr 2000) distearoylphosphatidylethanolamine followed by transfer into preformed liposomes during a one-step incubation. For cellular association, we used radiolabeled liposomes. Cytotoxicity was evaluated using the MTT in vitro proliferation assay., Results: The postinsertion approach to the formation of peptide-targeted liposomes led to the production of PLG bearing a maximum of approximately 0.3 microg antagonist G/micromol phospholipid. These liposomes had increased cellular association to H69 cells relative to nontargeted liposomes and, when loaded with doxorubicin, they resulted in similar levels of cytotoxicity to those obtained by conventional coupling techniques., Conclusions: The postinsertion technique is a simple, effective means for the production of biologically active peptide-targeted liposomes.

Chitsan (Ch) nanofiber mesh (NFM) is a material with natural characteristics favoring its use in human wound dressing. The present work proposes a gentamicin-loaded liposome immobilized at the surface of Ch NFMs to promote its antibacterial activity. To achieve this purpose, Ch NFMs were functionalized with thiol groups, and gentamicin-loaded liposomes were covalently immobilized by the reaction of the SH groups with maleimide. The maximum concentration of SH groups (55.52 ± 11.19 nmol cm −2 ) was obtained at pH 7. A fluorescent dye was covalently bound to the SH groups present at the surface of electrospun Ch NFMs. Their spatial distribution was uniform throughout the NFMs when analyzed by fluorescence microscopy. Gentamicin was successfully encapsulated into the liposomes with an efficiency of 17%. Gentamicin-loaded liposomes were uniformly distributed at the surface of the Ch NFMs and the drug release kinetic showed a sustained release of gentamicin during 16 h, achieving a steady state at 24 h. The in vitro susceptibility tests confirmed that the gentamicin released from the liposomes immobilized at the surface of electrospun Ch NFM has bactericidal activity against Escherichia coli , Pseudomonas aeruginosa and Staphylococcus aureus . The results show that the developed system has promising performance for wound dressing applications, avoiding infections caused by these common pathogens. [ABSTRACT FROM AUTHOR]

An overview of our experiences in the field of immunoliposomal anticancer drugs is provided with respect to choice of ligand, and choice of model system, in order to provide some guidance as to the rational use of this new technology. Liposomes targeted by either peptide or monoclonal antibodies showed significantly higher binding to their respective target cells in vitro compared to non-targeted liposomes in all model systems examined. This higher binding led to higher cytotoxicities relative to nontargeted liposomes. For the immunoliposomes to deliver their entrapped drug to target cell in vivo, long circulations half-lives are required. We have evaluated the pharmacokinetics of liposomes prepared by several different coupling techniques, and have found significant differences in the clearance of these immunoliposomes from the circulation. Immunoliposomes prepared with whole anti-CD19 IgG coupled by the Mal-PEGDSPE method demonstrated a short plasma half-life, which may reflect the random orientation of the MAb on the liposome surface. Coupling methods that mask or eliminate the Fc region result in immunoliposomes that have clearance rates more similar to untargeted liposomes. Insertion of peptides or antibodies into pre-formed liposomes through incubation with ligand-coupled PEG micelles resulted in immunoliposomes, termed post-insertion liposomes, that demonstrated comparable in vitro binding, pharmacokinetic profiles and in vivo therapeutic efficacy to liposomes made by conventional coupling methods. The therapeutic efficacy of liposomes, prepared by various coupling methods and targeted by different ligands, was compared in several different animal models of either haematological malignancies, pseudometastatic disease or solid tumours. In our hands, successful in vivo targeting has been obtained when the target is either small or readily accessible from the vasculature, where the liposomes have longer circulating half-lives and/or where a ligand against... [ABSTRACT FROM AUTHOR]

Abstract: We have previously described the development of novel sterically stabilized F3-targeted pH-sensitive liposomes, which exhibited the ability to target both cancer and endothelial cells. Herein, the therapeutic potential of those liposomes was assessed upon encapsulation of a siRNA against a well-validated molecular target, PLK1. Treatment of prostate cancer (PC3) and angiogenic endothelial (HMEC-1) cells with F3-targeted liposomes containing anti-PLK1 siRNA resulted in a significant decrease in cell viability, which was mediated by a marked PLK1 silencing, both at the mRNA and protein levels. Furthermore, pre-treatment of PC3 cells with F3-targeted liposomes containing anti-PLK1 siRNA enabled a 3-fold reduction of paclitaxel IC50 and a 2.5-fold augment of the percentage of cancer cells in G2/mitosis arrest, which ultimately culminated in cell death. Overall, the F3-targeted nanocarrier containing an anti-PLK1 siRNA might constitute a valuable system for prostate cancer treatment, either applied in a single schedule or combined with conventional chemotherapy. [Copyright &y& Elsevier]

[Display omitted] Ligand-mediated targeted liposomes have the potential to increase therapeutic efficacy of anticancer drugs. This work aimed to evaluate the ability of antagonist G, a peptide targeting agent capable of blocking the action of multiple neuropeptides, to selectivity improve targeting and internalization of liposomal formulations (long circulating liposomes, LCL, and stabilized antisense lipid particles containing ionizable amino lipid, SALP) to H69 and H82 small cell lung carcinoma (SCLC) cell lines. Antagonist G-targeted LCL and SALP were prepared by two different methods (either by direct covalent linkage at activated PEG grafted onto the liposomal surface or by post-insertion of DSPE-PEG-antagonist-G-conjugates into pre-formed liposomes). Association of the liposomal formulations with target SCLC cells was studied by fluorescence microscopy using fluorescence-labelled liposomes and confirmed quantitatively with [3H]-CHE-labelled liposomes. An antisense oligodeoxynucleotide against the overexpressed oncogene c-myc (as(c-myc)) was efficiently loaded into SALP formulations, the encapsulation efficiency decreased due to the inclusion of the targeting ligand. Also, liposome size was affected by as(c-myc) physical chemical properties. The amount of antagonist G linked to the surface of the liposomal formulations was dependent on the coupling method and lipid composition used. Covalent attachment of antagonist G increased liposomes cellular association and internalization via receptor-mediated and clathrin-dependent endocytosis, as assessed in SCLC cell lines. Biodistribution studies in healthy mice revealed a preferential lung accumulation of antagonist G–targeted SALP as compared to the non-targeted counterpart. Lung levels of the former were up to 3-fold higher 24 h after administration, highlighting their potential to be used as delivery vectors for SCLC treatment. [ABSTRACT FROM AUTHOR]