Long circulating liposomal platform utilizing hydrophilic polymer-based surface modification: preparation, characterisation, and biological evaluation.

Liposomes are one of the most important drug delivery vectors, nowadays used in clinics. In general, polyethylene glycol (PEG) is used to ensure the stealth properties of the liposomes. Here, we have employed hydrophilic, biocompatible and highly non-fouling N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers containing hydrophobic cholesterol anchors for the surface modification of liposomes, which were prepared by the method of lipid film hydration and extrusion through 100 nm polycarbonate filters. Efficient surface modification of liposomes was confirmed by transmission electron microscopy, atomic force microscopy, and gradient ultracentrifugation. The ability of long-term circulation in the vascular bed was demonstrated in rabbits after i.v. application of fluorescently labelled liposomes. Compared to PEGylated liposomes, HPMA-based copolymer-modified liposomes did not induce specific antibody formation and did not activate murine and human complement. Compared with PEGylated liposomes, HPMA-based copolymer-modified liposomes showed a better long-circulating effect after repeated administration. HPMA-based copolymer-modified liposomes thus represent suitable new candidates for a generation of safer and improved liposomal drug delivery platforms.

Blood vessel wall shear stress determines regions of liposome accumulation in angiogenic vasculature.

Nanoparticles used for drug delivery often require intravenous administration exposing them to fluid forces within the vasculature, yet the impact of blood flow on nanoparticle delivery remains incompletely understood. Here, we utilized transgenic zebrafish embryos to investigate the relationship between the accumulation of fluorescently labeled PEGylated liposomes and various hemodynamic factors (such as flow velocity, wall shear stress (WSS), and flow pattern) across a wide range of angiogenic blood vessels. We reconstructed 3D models of vascular structures from confocal images and used computational fluid dynamics to calculate local WSS, velocities, and define flow patterns. The spatial distribution of fluorescently labeled liposomes was subsequently mapped within the same 3D space and correlated with local hemodynamic parameters. Through the integration of computational fluid dynamics and in vivo experimentation, we show that liposomes accumulated in vessel regions with WSS between 0.1-0.8 Pa, displaying an inverse linear correlation (R2 > 0.85) between time-averaged wall shear stress and liposome localization in vivo. Interestingly, flow pattern did not appear to impact liposome accumulation. Collectively, our findings suggest the potential of stealth liposomes for passive targeting of low-flow vasculature, including capillaries and intricate angiogenic vasculature resembling that of tumor vessel networks.

Inclusion Complex between Local Anesthetic/2-hydroxypropyl-ß-cyclodextrin in Stealth Liposome.

The drugs delivery system in the treatment of diseases has advantages such as reduced toxicity, increased availability of the drug, etc. Therefore, studies of the supramolecular interactions between local anesthetics (LAs) butamben (BTB) or ropivacaine (RVC) complexed with 2-hydroxypropyl-ß-cyclodextrin (HP-ßCD) and carried in Stealth liposomal (SL) are performed. 1H-NMR nuclear magnetic resonance (DOSY and STD) were used as the main tools. The displacements observed in the 1H-NMR presented the complexion between LAs and HP-ßCD. The diffusion coefficients of free BTB and RVC were 7.70 × 10-10 m2 s-1 and 4.07 × 10-10 m2 s-1, and in the complex with HP-ßCD were 1.90 × 10-10 m2 s-1 and 3.64 × 10-10 m2 s-1, respectively, which indicate a strong interaction between the BTB molecule and HP-ßCD (98.3% molar fraction and Ka = 72.279 L/mol). With STD-NMR, the encapsulation of the BTB/HP-ßCD and RVC/HP-ßCD in SL vesicles was proven. Beyond the saturation transfer to the LAs, there is the magnetization transfer to the hydrogens of HP-ßCD. BTB and RVC have already been studied in normal liposome systems; however, little is known of their behavior in SL.

Liposomes: structure, composition, types, and clinical applications.

Liposomes are now considered the most commonly used nanocarriers for various potentially active hydrophobic and hydrophilic molecules due to their high biocompatibility, biodegradability, and low immunogenicity. Liposomes also proved to enhance drug solubility and controlled distribution, as well as their capacity for surface modifications for targeted, prolonged, and sustained release. Based on the composition, liposomes can be considered to have evolved from conventional, long-circulating, targeted, and immune-liposomes to stimuli-responsive and actively targeted liposomes. Many liposomal-based drug delivery systems are currently clinically approved to treat several diseases, such as cancer, fungal and viral infections; more liposomes have reached advanced phases in clinical trials. This review describes liposomes structure, composition, preparation methods, and clinical applications.

Lipid-Based Nanoparticle Formulation of Diallyl Trisulfide Chemosensitizes the Growth Inhibitory Activity of Doxorubicin in Colorectal Cancer Model: A Novel In Vitro, In Vivo and In Silico Analysis.

Garlic's main bioactive organosulfur component, diallyl trisulfide (DATS), has been widely investigated in cancer models. However, DATS is not suitable for clinical use due to its low solubility. The current study seeks to improve DATS bioavailability and assess its chemopreventive and chemosensitizing properties in an AOM-induced colorectal cancer model. The polyethylene glycol coated Distearoylphosphatidylcholine/Cholesterol (DSPC/Chol) comprising DATS-loaded DATSL and doxorubicin (DOXO)-encapsulated DOXL liposomes was prepared and characterized. The changes in the sensitivity of DATS and DOXO by DATSL and DOXL were evaluated in RKO and HT-29 colon cancer cells. The synergistic effect of DATSL and DOXL was studied by cell proliferation assay in the combinations of IC10, IC25, and IC35 of DATSL with the IC10 of DOXL. AOM, DATSL, and DOXL were administered to different groups of mice for a period of 21 weeks. The data exhibited ~93% and ~46% entrapment efficiency of DATSL and DOXL, respectively. The size of sham liposomes was 110.5 nm, whereas DATSL and DOXL were 135.5 nm and 169 nm, respectively. DATSL and DOXL exhibited significant sensitivity in the cell proliferation experiment, lowering their IC50 doses by more than 8- and 14-fold, respectively. However, the DATSL IC10, IC25, and IC35 showed escalating chemosensitivity, and treated the cells in combination with DOXL IC10. Analysis of histopathological, cancer marker enzymes, and antioxidant enzymes revealed that the high dose of DATSL pretreatment and DOXL chemotherapy is highly effective in inhibiting AOM-induced colon cancer promotion. The combination of DATSL and DOXL indicated promise as a colorectal cancer treatment in this study. Intermolecular interactions of DATS and DOXO against numerous cancer targets by molecular docking indicated MMP-9 as the most favourable target for DATS exhibiting binding energy of -4.6 kcal/mol. So far, this is the first research to demonstrate the chemopreventive as well as chemosensitizing potential of DATSL in an animal model of colorectal cancer.

The Effect of Liposomal Diallyl Disulfide and Oxaliplatin on Proliferation of Colorectal Cancer Cells: In Vitro and In Silico Analysis.

Diallyl disulfide (DADS) is one of the main bioactive organosulfur compounds of garlic, and its potential against various cancer models has been demonstrated. The poor solubility of DADS in aqueous solutions limits its uses in clinical application. The present study aimed to develop a novel formulation of DADS to increase its bioavailability and therapeutic potential and evaluate its role in combination with oxaliplatin (OXA) in the colorectal cancer system. We prepared and characterized PEGylated, DADS (DCPDD), and OXA (DCPDO) liposomes. The anticancer potential of these formulations was then evaluated in HCT116 and RKO colon cancer cells by different cellular assays. Further, a molecular docking-based computational analysis was conducted to determine the probable binding interactions of DADS and OXA. The results revealed the size of the DCPDD and DCPDO to be 114.46 nm (95% EE) and 149.45 nm (54% EE), respectively. They increased the sensitivity of the cells and reduced the IC50 several folds, while the combinations of them showed a synergistic effect and induced apoptosis by 55% in the cells. The molecular docking data projected several possible targets of DADS and OXA that could be evaluated more precisely by these novel formulations in detail. This study will direct the usage of DCPDD to augment the therapeutic potential of DCPDO against colon cancer in clinical settings.

Stealth Liposomes (PEGylated) Containing an Anticancer Drug Camptothecin: In Vitro Characterization and In Vivo Pharmacokinetic and Tissue Distribution Study.

Numerous attempts to overcome the poor water solubility of cam ptothecin (CPT) by various nano drug delivery systems are described in various sources in the literature. However, the results of these approaches may be hampered by the incomplete separation of free CPT from the formulations, and this issue has not been investigated in detail. This study aimed to promote the solubility and continuous delivery of CPT by developing long-lasting liposomes using various weights (M.W. 2000 and 5000 Daltons) of the hydrophilic polymer polyethylene glycol (PEG). Conventional and PEGylated liposomes containing CPT were formulated via the lipid film hydration method (solvent evaporation) using a rotary flash evaporator after optimising various formulation parameters. The following physicochemical characteristics were investigated: surface morphology, particle size, encapsulation efficiency, in vitro release, and formulation stability. Different molecular weights of PEG were used to improve the encapsulation efficiency and particle size. The stealth liposomes prepared with PEG5000 were discrete in shape and with a higher encapsulation efficiency (83 ± 0.4%) and a prolonged rate of drug release (32.2% in 9 h) compared with conventional liposomes (64.8 ± 0.8% and 52.4%, respectively) and stealth liposomes containing PEG2000 (79.00 ± 0.4% and 45.3%, respectively). Furthermore, the stealth liposomes prepared with PEG5000 were highly stable at refrigeration temperature. Significant changes were observed using various pharmacokinetic parameters (mean residence time (MRT), half-life, elimination rate, volume of distribution, clearance, and area under the curve) of stealth liposomes containing PEG2000 and PEG5000 compared with conventional liposomes. The stealth liposomes prepared with PEG5000 showed promising results with a slow rate of release over a long period compared with conventional liposomes and liposomes prepared with PEG2000, with altered tissue distribution and pharmacokinetic parameters.

Liposome Photosensitizer Formulations for Effective Cancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising non-invasive strategy in the fight against that which circumvents the systemic toxic effects of chemotherapeutics. It relies on photosensitizers (PSs), which are photoactivated by light irradiation and interaction with molecular oxygen. This generates highly reactive oxygen species (such as 1O2, H2O2, O2, ·OH), which kill cancer cells by necrosis or apoptosis. Despite the promising effects of PDT in cancer treatment, it still suffers from several shortcomings, such as poor biodistribution of hydrophobic PSs, low cellular uptake, and low efficacy in treating bulky or deep tumors. Hence, various nanoplatforms have been developed to increase PDT treatment effectiveness and minimize off-target adverse effects. Liposomes showed great potential in accommodating different PSs, chemotherapeutic drugs, and other therapeutically active molecules. Here, we review the state-of-the-art in encapsulating PSs alone or combined with other chemotherapeutic drugs into liposomes for effective tumor PDT.

Targeted Liposomes Encapsulating miR-603 Complexes Enhance Radiation Sensitivity of Patient-Derived Glioblastoma Stem-Like Cells.

Despite potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a strategy for miRNA delivery in the treatment of glioblastoma, the most common form of adult brain cancer, that involves complexation of miRNA with polyethylenimine (PEI) and encapsulation in targeted liposomes. miRNA 603 (miR-603) is a master regulatory miRNA that suppresses glioblastoma radiation resistance through down-regulation of insulin-like growth factor 1 (IGF1) signaling. miR-603 was complexed with PEI, a cationic polymer, and encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5ß1 integrin that is overexpressed in glioblastomas. Cultured patient-derived glioblastoma cells internalized PR_b-functionalized liposomes but not the non-targeted liposomes. The integrin targeting and complexation of the miRNA with PEI were associated with a 22-fold increase in intracellular miR-603 levels, and corresponding decreases in IGF1 and IGF1 receptor (IGF1R) mRNA expression. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI sensitized the cells to ionizing radiation (IR), a standard of care treatment for glioblastomas. These results suggest that PR_b-functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a therapeutic platform for glioblastomas.

Targeted PD-L1 PLGA/liposomes-mediated luteolin therapy for effective liver cancer cell treatment.

Stealth PLGA/Liposome nanoparticles (NPs) modified with tumor-targeting PD-L1 antibody for systemic delivery of luteolin for liver cancer were prepared. The morphologies and therapeutic effects of luteolin-loaded PD-L1 targeted stealth PLGA/Liposomes (L-PD-SP/Ls) in vitro were analyzed. Functional L-PD-P/L NPs composed of PLGA, DOPC and DSPE-PEG display low cell cytoxicity in HepG2 cells, and has more cell uptake ability than P/Ls NPs. L-PD-SP/Ls was more effective in inhibiting HepG2 cell proliferation than free luteolin in solution (p < 0.05) and luteolin-loaded P/Ls (p < 0.05). Compared with the cell control group and the non-PD-L1 targeted group, the mediated effect of PD-L1 can significantly enhance the uptake of drugs by cells, and L-PD-SP/Ls can significantly reduce the expression of Bcl-2 and increase the level of LDH in cells. Our findings collectively support the utility of PD-L1-targeted P/L NPs as a potentially effective drug delivery system.

Fluorescence-based method for sensitive and rapid estimation of chlorin e6 in stealth liposomes for photodynamic therapy against cancer.

Chlorin e6 is a chlorine-based porphyrin containing photosensitizer mainly used for the therapy in cancers like neck and head, early-stage lung cancer, and topical skin cancers. The present study provides a comprehensive account of a highly sensitive, precise, and validated method for the quantification of chlorin e6 in its liposomal formulation. This method is based on the systemic study of the fluorescence action of chlorin e6 in acetonitrile solvent. This experiment follows the analytical method validation parameters as per the International Conference on Harmonization (ICH). Chlorin e6 molecule exhibits strong fluorescence at a wavelength of emission 665 nm, upon excitation at a wavelength of excitation 400 nm in acetonitrile. The linearity of the fluorescence concentration plot was observed over a concentration range of 50 to 1000 ng/mL. The developed and validated method was successfully applied for the estimation of encapsulation efficiency in in-house developed stealth liposomes. Also, stock solution stability and photodegradation study of chlorin e6 were further conducted.

Bulk and Microfluidic Synthesis of Stealth and Cationic Liposomes for Gene Delivery Applications.

This chapter describes the synthesis of stealth and cationic liposomes and their complexation with plasmid DNA to generate lipoplexes for gene delivery applications. Two techniques are presented: a top-down approach which requires a second step of processing for downsizing the liposomes (i.e., ethanol injection method) and a microfluidic technique that explores the diffusion of ethanol in water to allow the proper lipid self-assembly. The synthesis of stealth liposomes is also a challenge since the use of poly(ethylene glycol) favors the formation of oblate micelles. In this protocol, the stealth cationic liposome synthesis by exploring the high ionic strength to overcome the formation of secondary structures like micelles is described. Finally, the electrostatic complexation between cationic liposomes and DNA is described, indicating important aspects that guarantee the formation of uniform lipoplexes.

Supercritical fluid assembly of albendazole liposomes targeting gastrin-releasing peptide receptor overexpressing tumors.

Aim: To develop albendazole (ABZ)-loaded bombesin(6-14) (BBN(6-14)) functionalized liposomes for targeting GRPR to enhance delivery to cancer cells. Materials & methods: ABZ-loaded liposomes were formulated using supercritical CO2 technology; functionalized with a GRPR-targeted lipid-anchored BBN(6-14) peptide; and evaluated for effects on cell viability, particle size and targeted cell uptake. Results: BBN(6-14)-coated ABZ liposomes decreased cell viability compared with nonfunctionalized ABZ liposomes. The level of GRPR expression positively correlated with intracellular uptake and decreased cell viability. The reduced cell viability, higher cell uptake and GRPR expression were observed in the order PC-3 > Caco-2 > HepG2 cells. Conclusion: BBN(6-14)-functionalized ABZ liposomes showed enhanced reduction in cell viability compared with nonfunctionalized ABZ liposomes.

Biocompatible Nanovesicular Drug Delivery Systems with Targeting Potential for Autoimmune Diseases.

Autoimmune diseases are collectively addressed as chronic conditions initiated by the loss of one's immunological tolerance, where the body treats its own cells as foreigners or self-antigens. These hay-wired antibodies or immunologically capable cells lead to a variety of disorders like rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, multiple sclerosis and recently included neurodegenerative diseases like Alzheimer's, Parkinsonism and testicular cancer triggered T-cells induced autoimmune response in testes and brain. Conventional treatments for autoimmune diseases possess several downsides due to unfavourable pharmacokinetic behaviour of drug, reflected by low bioavailability, rapid clearance, offsite toxicity, restricted targeting ability and poor therapeutic outcomes. Novel nanovesicular drug delivery systems including liposomes, niosomes, proniosomes, ethosomes, transferosomes, pharmacosomes, ufasomes and biologically originated exosomes have proved to possess alluring prospects in supporting the combat against autoimmune diseases. These nanovesicles have revitalized available treatment modalities as they are biocompatible, biodegradable, less immunogenic and capable of carrying high drug payloads to deliver both hydrophilic as well as lipophilic drugs to specific sites via passive or active targeting. Due to their unique surface chemistry, they can be decorated with physiological or synthetic ligands to target specific receptors overexpressed in different autoimmune diseases and can even cross the blood-brain barrier. This review presents exhaustive yet concise information on the potential of various nanovesicular systems as drug carriers in improving the overall therapeutic efficiency of the dosage regimen for various autoimmune diseases. The role of endogenous exosomes as biomarkers in the diagnosis and prognosis of autoimmune diseases along with monitoring progress of treatment will also be highlighted.

In Vitro and in Vivo Behavior of Liposomes Decorated with PEGs with Different Chemical Features.

The colloidal stability, in vitro toxicity, cell association, and in vivo pharmacokinetic behavior of liposomes decorated with monomethoxy-poly(ethylene glycol)-lipids (mPEG-lipids) with different chemical features were comparatively investigated. Structural differences of the mPEG-lipids used in the study included: (a) surface-anchoring moiety [1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cholesterol (Chol), and cholane (Chln)]; (b) mPEG molecular weight (2 kDa mPEG45 and 5 kDa mPEG114); and (c) mPEG shape (linear and branched PEG). In vitro results demonstrated that branched (mPEG114)2-DSPE confers the highest stealth properties to liposomes (∼31-fold lower cell association than naked liposomes) with respect to all PEGylating agents tested. However, the pharmacokinetic studies showed that the use of cholesterol as anchoring group yields PEGylated liposomes with longer permeance in the circulation and higher systemic bioavailability among the tested formulations. Liposomes decorated with mPEG114-Chol had 3.2- and ∼2.1-fold higher area under curve (AUC) than naked liposomes and branched (mPEG114)2-DSPE-coated liposomes, respectively, which reflects the high stability of this coating agent. By comparing the PEGylating agents with same size, namely, linear 5 kDa PEG derivatives, linear mPEG114-DSPE yielded coated liposomes with the best in vitro stealth performance. Nevertheless, the in vivo AUC of liposomes decorated with linear mPEG114-DSPE was lower than that obtained with liposomes decorated with linear mPEG114-Chol. Computational molecular dynamics modeling provided additional insights that complement the experimental results.

Ionic strength for tailoring the synthesis of monomodal stealth cationic liposomes in microfluidic devices.

In this work, we describe a hydrodynamic flow-focusing microfluidic process to produce stealth cationic liposomes (SCL), stabilized with poly(ethylene glycol) (PEG), with uniform and reproducible features. Through cryogenic transmission electron microscopy (cryo-TEM) characterization and real-time monitoring, we verified the formation of multi-sized lipid self-aggregates, which can be attributed to micelles formation. These structures tend to undergo deposition within the PDMS/glass microchannels through intermolecular interactions with the glass walls, hindering not only the process reproducibility but also the final biological application of the SCL products. In view of this, we propose the modulation of ionic strength of the side streams aiming to ionically shield the glass surface, decrease the intermolecular interactions of the lipid polar heads, and, essentially, to promote the bilayer-driven self-assembly of SCL with 1% of DSPE-PEG2000 lipid. Herein, we applied phosphate-buffered saline (PBS) from 10 to 50 mM concentration as side streams, and evaluated its effects on SCL final physicochemical properties in terms of size distribution, mean diameter, zeta potential and polydispersity index (PDI). We present evidences indicating that the ionic strength can be used as a microfluidic process parameter to modulate the lipids self-assembly kinetics whilst preventing micelles formation. Finally, the proposed diffusion-based microfluidic system with high ionic strength enables the formation of monodisperse (PDI < 0.2) SCL of around 140 nm with monomodal size distributions and enhanced properties when compared to usual bulk mixing.

Physicochemical study of the protein-liposome interactions: influence of liposome composition and concentration on protein binding.

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (Fs), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when Fs is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.

Interaction between C18 fatty acids and DOPE PEG2000 in Langmuir monolayers: effect of degree of unsaturation.

In this study, we address the effect of the cis-double bond in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamide-N-[methoxy(polyethylene glycol)-2000, DOPE PEG2000 (DP), on the Langmuir monolayer of C18 fatty acids-namely, stearic acid (SA), oleic acid (L1), linoleic acid (L2), and linolenic acid (L3)-with the same head group but different degrees of saturation on their hydrocarbon chains. Negative values of Gibbs free energy of mixing (ΔG mix) were obtained throughout the investigated ranges of the unsaturated C18 fatty-acid (L1, L2 and L3) mixed systems, indicating that very strong attractions occurred between molecules in the monolayers. The bend and kink effects from the cis-double bond(s) in the hydrocarbon chain affected the membrane fluidity and molecular packing in the monolayers, which resulted in a greater interaction between unsaturated C18 fatty acids and DP. The most thermodynamically stable mole composition of unsaturated C18 fatty acids to DP was observed at 50:1; this ratio is suggested to be the best mole ratio and will be subsequently used to prepare DP-C18 fatty-acid nanoliposomes. The presence of cis-double bonds in both hydrocarbon chains of DOPE in DP also created an imperfection in the membrane structure of lipid-drug delivery systems, which is expected to enhance lipid-based systems for antibody conjugation and drug encapsulation.

Interaction between PEG lipid and DSPE/DSPC phospholipids: An insight of PEGylation degree and kinetics of de-PEGylation.

The degree to which liposomes are PEGylated is the feature, which most influences the length of the presence of stealth liposomes in the bloodstream. In order to thoroughly investigate the maximum amount of DSPE-PEG2000 that can be used to stabilize stealth liposomes, these were synthesized at different concentrations of DSPE-PEG2000 and their physicochemical properties were investigated by using differential scanning calorimetry (DSC). The kinetics of PEGylation and de-PEGylation were performed by incubating non-stealth liposomes in a DSPE-PEG2000 suspension at different incubation times, and then analyzing the data using DSC and dynamic light scattering (DLS) techniques. The results demonstrated that DSPE-PEG2000 was self-assembled in the phospholipid bilayers, thus forming stealth liposomes. The different amounts of DSPE-PEG2000 in the bilayer triggered a de-PEGylation phenomenon, resulting in mixed nanoaggregates, which derived from the detergent-like properties of the PEGylated phospholipids.

Dual-ligand α5ß1 and α6ß4 integrin targeting enhances gene delivery and selectivity to cancer cells.

Nanoparticles functionalized with cancer-targeting ligands have shown promise but are still limited by off-tumor binding to healthy tissues that express low levels of the molecular target. Targeting two cancer biomarkers using dual-targeted heteromultivalent nanoparticles presents a possible solution to this challenge by requiring overexpression of two separate ligands for localization. In order to guide experimental design, a kinetic model was built to explore how the affinity and valency of dual-ligand liposomes affect the binding and selectivity of delivery to cells with various receptor expression. α5ß1 and α6ß4 integrin expression levels were quantified on 20 different cell lines to identify appropriate model cells for in vitro investigation. Dual-targeting heteromultivalent liposomes covered with polyethylene glycol (PEG) were synthesized using the PR_b peptide that binds to the α5ß1 integrin and the AG86 peptide that binds to the α6ß4 integrin. PEGylated liposomes with varying ratios of the targeting peptides were delivered to cells with different integrin concentrations. Nanoparticle binding and internalization as well as integrin internalization as a function of time were evaluated to understand the effect of valency and avidity on delivery. Results showed that of all formulations and cells tested, dual-ligand liposomes with equal ligand valencies achieved enhanced binding and selectivity for cancer cells expressing equal and high levels of receptor expression. These trends were consistent between theoretical and experimental results. The optimized liposomes were further used to achieve efficient and selective transfection in dual-receptor expressing cancer cells. With a quantitative understanding of dual-ligand liposome binding, the insights gained from this study can inform rational design of modular heteromultivalent nanoparticles for enhanced specificity to target tissue for the creation of more effective cancer treatments.