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Extracellular vesicles (EVs) are future promising therapeutics, but their instability in vivo after administration remains an important barrier to their further development. Many groups evaluated EV surface modification strategies to add a targeting group with the aim of controlling EV biodistribution. Conversely, fewer groups focused on their stabilization to obtain "stealth" allogenic EVs. Modulating their stabilization and biodistribution is an essential prerequisite for their development as nano-therapeutics. Here, we explored polyoxazolines with lipid anchors association to the EV membrane (POxylation as an alternative to PEGylation) to stabilize EVs in plasma and control their biodistribution, while preserving their native properties. We found that this modification maintained and seemed to potentiate the immunomodulatory properties of EVs derived from mesenchymal stem/stromal cells (MSC). Using a radiolabeling protocol to track EVs at a therapeutically relevant concentration in vivo , we demonstrated that POxylation is a promising option to stabilize EVs in plasma because it increased EV half-life by 6 fold at 6 h post-injection. Moreover, EV accumulation in tumors was higher after POxylation than after PEGylation. [Display omitted] • Preservation of mouse MSC-EV native immunomodulatory properties after POxylation. • Radiolabeling protocol allowed EV detection after intravenous administration of 1.108 EVs/mouse until 24h. • POxylated EVs showed enhanced plasma stabilization after intravenous administration in mice. • Higher accumulation in PANC-1 cell tumors of POxylated EVs than PEGylated EVs. [ABSTRACT FROM AUTHOR]

Objective(s): Brain cancer treatments have mainly failed due to their inability to cross the blood-brain barrier. Several studies have confirmed the presence of glutathione (GSH) receptors on BBB’s surface, as a result, products like 2B3-101, which contain over 5% pre-inserted GSH PEGylated liposomal Doxorubicin, are being tested in clinical trials. Here we conducted the PEGylated nanoliposomal Doxorubicin particles that are covalently attached to the glutathione using the post-insertion technique. Compared with the pre-insertion approach, the post-insertion method is notably simpler, faster, and more cost-effective, making it ideal for large-scale pharmaceutical manufacturing. Materials and Methods: The ligands of the DSPE PEG(2000) Maleimide-GSH were introduced in the amounts of 25, 50, 100, 200, and 400 on the available Caelyx. Following physicochemical evaluations, animal experiments such as biodistribution, fluorescence microscopy, and pharmacokinetics were done. Results: In comparison with Caelyx, the 200L and 400L treatment arms were the most promising formulations. We showed that nanocarriers containing 40 times fewer GSH micelles than 2B3-101 significantly increased blood-brain barrier penetrance. Due to the expressed GSH receptors on tissues as an endogenous antioxidant, Doxorubicin will likely concentrate in the liver, spleen, heart, and lung in comparison with Caelyx, according to other tissue analyses. Conclusion: The post-insertion technique was found a successful approach with more pharmaceutical aspects for large-scale production. Moreover, further investigations are highly recommended to determine the efficacy of 5% post-inserted GSH targeted nanoliposomes versus 2B3-101 as a similar formulation with a different preparation method.

Objective(s): Brain cancer treatments have mainly failed due to their inability to cross the bloodbrain barrier. Several studies have confirmed the presence of glutathione (GSH) receptors on BBB's surface, as a result, products like 2B3-101, which contain over 5% pre-inserted GSH PEGylated liposomal doxorubicin, are being tested in clinical trials. Here we conducted the PEGylated nanoliposomal doxorubicin particles that are covalently attached to the glutathione using the postinsertion technique. Compared with the pre-insertion approach, the post-insertion method is notably simpler, faster, and more cost-effective, making it ideal for large-scale pharmaceutical manufacturing. Materials and Methods: The ligands of the DSPE PEG(2000) Maleimide-GSH were introduced in the amounts of 25, 50, 100, 200, and 400 on the available Caelyx. Following physicochemical evaluations, animal experiments such as biodistribution, fluorescence microscopy, and pharmacokinetics were done. Results: In comparison with Caelyx, the 200L and 400L treatment arms were the most promising formulations. We showed that nanocarriers containing 40 times fewer GSH micelles than 2B3-101 significantly increased blood-brain barrier penetrance. Due to the expressed GSH receptors on tissues as an endogenous antioxidant, doxorubicin will likely concentrate in the liver, spleen, heart, and lung in comparison with Caelyx, according to other tissue analyses. Conclusion: The post-insertion technique was found a successful approach with more pharmaceutical aspects for large-scale production. Moreover, further investigations are highly recommended to determine the efficacy of 5% post-inserted GSH targeted nanoliposomes versus 2B3-101 as a similar formulation with a different preparation method. [ABSTRACT FROM AUTHOR]

PEGylated liposomes are one of the useful boron carriers for boron neutron capture therapy (BNCT). Recently, a method of adding PEG after liposome formation (post-insertion) was reported. In this study, we prepared polyborane-encapsulated PEGylated liposomes for BNCT with half the amount of DSPE-PEG of the conventional method using post-insertion technique (post-PEG liposomes), and their usefulness were evaluated in comparison with conventional PEGylated liposomes (pre-PEG liposomes). From the results of physicochemical property measurements, it was confirmed that particle size distributions, surface charge densities, and fixed aqueous layer thicknesses of these liposomes were equivalent. In vitro cytotoxicity and cell uptake tests were also carried out using B16 melanoma and RAW264.7 cells. Polyborane solution and bare liposomes were used for comparison. From the results of these tests, we confirmed that post-PEG liposomes and pre-PEG liposomes have the same influence of PEGylation. To evaluate biodistribution properties at 24 h post-administration, these liposomes and polyborane solution were injected into the tail veins of tumor-bearing mice. Boron concentration and tumor/blood ratios of PEGylated liposomes were 73.2-77.6 µg/g of tumor tissue and 5.5-5.8, respectively. From these results, it was found that by using post-insertion technique, liposomes for BNCT having same effect as the liposome prepared using the conventional method can be prepared with half amount of DSPE-PEG. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Phosphatidyl-choline liposomes can be PEGylated by post-insertion technique. • It avoids the hindrance of the aqueous compartments elicited by the PEG moieties. • The concentration of cholesterol is proportional to the retention of PEG-derivative. • A plateau of insertion is obtained at a concentration of PEG-derivative ≥10% w/w. Abstract The insertion of specific derivatives into pre-formed colloidal systems has been shown to be a useful method for modifying their pharmacokinetic characteristics and biodistribution profiles. In this experimental work the effect of the post-insertion of different PEG-derivatives into pre-formed 100-nm liposomes made up of various lipid mixtures (DMPC, DPPC, DOPC, DSPC and cholesterol at different molar ratios) was investigated. The vesicles were incubated with increasing amounts of DSPE-mPEG 2000 as sterically stabilized micelles (5, 10 and 15% w/w with respect to the liposomal lipid mixture) in order to favour the insertion of the PEG-lipid into the liposomal bilayer. The colloidal formulations were characterized by photo-correlation spectroscopy; the DSPE-mPEG 2000 integrated into the pre-formed liposomes was demonstrated by means of field flow fractionation while the amount of post-inserted compound was quantified using a suitable spectrophotometric assay (I 2 assay). Similar investigations have been performed using PEG-derivatives characterized by a different molecular weight. The physico-chemical properties of the various liposomal formulations were influenced by the post-insertion of PEG-derivatives. The lipid mixture made up of saturated phospholipids and cholesterol proved to be the best, post-insertion (P.I.E.). The post-insertion technique may be a suitable approach to be used in personalized (nano)medicine. [ABSTRACT FROM AUTHOR]

Hyaluronan (MW: 1.5 MDa) was linked to a phospholipid (dipalmitoyl phosphatidylethanolamine, DPPE) by an amidification procedure to obtain novel macromolecules (HA-DPPE) able to coat liposomes. Liposomes made of dipalmitoyl phosphatidylcholine and cholesterol (DPPC/Chol: 95/5 molar ratio), with a mean size around 100 nm, were incubated with HA-DPPE at 55 °C, allowing the insertion of DPPE moieties in the liposomal bilayer and leading to hyaluronan-coated liposomes (HAsomes) as evidenced by several techniques including dynamic light scattering and differential scanning calorimetry. The amount or HA-DPPE coating liposomes was quantified by different methods among which capillary electrophoresis and their stability in serum was finally compared to that of plain liposomes. As a conclusion, we provide insight into the physico-chemical characterization of HA-DPPE and of HAsomes demonstrating that easy coating of phospholipid vesicles can be achieved by post-insertion of a lipid derivative of hyaluronan. This approach represents an innovative strategy for coating vesicular systems to confer them simultaneously with long circulation properties and selective targeting towards HA-receptors. [ABSTRACT FROM AUTHOR]

Purpose: The in vitro and in vivo properties of PEGylated pH-sensitive liposomes (PSL) prepared by pre- and post-insertion techniques were investigated. Methods: A pre-insertion or post-insertion technique was used for PSL PEGylation. For the first time, confocal laser scanning microscopy coupled with a modified calcein self-quench assay was applied to evaluate the endosome escape capability. PSL cellular uptake was evaluated using macrophages and the cytotoxicity using a gemcitabine (model drug)-resistant MIA PaCa-2 cells. The pharmacokinetics of PSL encapsulated gemcitabine was investigated in rats. Results: PEGylation reduced the pH-sensitivity in a concentration-dependent manner (0.5-5% mol). Both PEGylation methods reduced the uptake of PSL by macrophages by over 60%. Cytotoxicity was ranked in the order: post-inserted PSL ≥ pre-inserted PSL > non-PSL > gemcitabine solution, consistent with the confocal microscopic observation and pH-sensitivity. Both pre and post-inserted PSL resulted in significant reductions ( p < 0.05) in plasma clearance (58.6 and 38.4 ml/h/kg), increases in the area-under-the-concentration-time curve (56.9 and 87.1 μM· h) and half-life (6.1 and 6.2 h) compared to gemcitabine solution (152.9 ml/h/kg, 22.2 μM· h and 1.4 h). Conclusion: PEGylation by post-insertion offers advantages over pre-insertion to obtain PSL with enhanced pH-sensitivity, more effective intra-cytoplasmic delivery, and a superior pharmacokinetics. [ABSTRACT FROM AUTHOR]

Abstract: Attaching polysaccharides to the surface of nanoparticles offers the possibility of modifying the physicochemical and biological properties of the core particles. The surface of lipid nanocapsules (LNCs) was modified by post-insertion of amphiphilic lipochitosan (LC) or lipodextran (LD). Modelling of these LNCs by the drop tensiometer technique revealed that the positively charged LC made the LNC surface more rigid, whereas the neutral, higher M W LD had no effect on the surface elasticity. Both LNC-LC and LNC-LD activated the complement system more than the blank LNC, thus suggesting increased capture by the mononuclear phagocyte system. In vitro, the positively charged LNC-LC were more efficiently bound by the model HEK293(β3) cells compared to LNC and LNC-LD. Finally, it was observed that neither LC nor LD changed the in vivo biodistribution properties of LNCs in mice. These polysaccharide-coated LNCs, especially LNC-LC, are promising templates for targeting ligands (e.g. peptides, proteins) or therapeutic molecules (e.g. siRNA). [Copyright &y& Elsevier]

Abstract: We report synthesis and characterization of a novel PEG2000-conjugated hexadecylcarbamoylmethyl hexadecanoate (HDAS-PEG) as a PEG-phospholipid substitute for enhancing circulation persistence of liposomes. HDAS-PEG showed critical micelle concentration of 4.25μM. We used post-insertion technique to introduce HDAS-PEG in outer lipid layer of the preformed liposomes. The presence of surface HDAS-PEG was confirmed by altered electrophoretic mobility, confocal microscopy and PEG estimation by ELISA. The post-inserted HDAS-PEG desorbed at approximately half the rate at which post-inserted DSPE-PEG desorbed from the liposome surface. HDAS-PEG significantly reduced liposome-induced complement activation (C4d, Bb and SC5b); HDAS-PEG was more effective than more commonly used DSPE-PEG in this capacity. For studying circulation persistence, the liposomes were labeled with 99mTc radionuclide and administered in rats. 99mTc-HDAS-PEG-liposomes showed prolonged persistence in blood as compared to that shown by 99mTc-plain liposomes. After 24h of administration, <1% of 99mTc-plain liposomes remained in blood, whereas approximately 28% of injected 99mTc-HDAS-PEG-liposomes were present in blood. In comparison, only 4.8% of 99mTc-DSPE-PEG-liposomes were measured in blood after 24h. As expected, the clearance route of the liposomes was through liver and spleen. These results demonstrate the potential of a novel non-phosphoryl HDAS-PEG for surface modification of preformed liposomes with a goal of prolonging their circulation persistence and more effective inhibition of complement activation. [Copyright &y& Elsevier]

Abstract: Lipidic NanoCapsules (LNCs) were prepared via an emulsion phase inversion method. Nanoparticles with hydrodynamic diameter of 25, 50 and 100nm were easily obtained. Their surfaces are covered with short PEG chains (PEG 660) which are not bearing any chemical reactivities. Thus, in order to overcome this handicap towards post-functionalization possibilities, post-insertion of DSPE-PEG2000 amino (DSPA) can be employed. In order to characterize the insertion step, we have developed a chemical assay for the quantification of amino group inside the PEG shell of LNCs. Subsequently, the post-insertion yield was found to be comprised between 60 and 90% whatever the hydrodynamic diameter of the LNCs is. By means of simple calculations, the density of amino group is estimated to be closed to 0.2 and 1.2molecules/nm2. The formulation of LNCs and their controlled functionalization represent an interesting system for the development of bionanoconjugates in a short and effective process. [Copyright &y& Elsevier]

Abstract: Lipid nanocapsules (LNCs) were modified by adsorbing sequentially dextran sulfate (DS) and chitosan (CS) on their surface by the layer-by-layer (LBL) approach. Tangential flow filtration (TFF) was used in intermediate purifications of the LNC dispersion during the LBL process. The surface modification was based on electrostatic interactions between the coating polyelectrolytes (PEs) and the LNCs. Therefore, a cationic surfactant, lipochitosan (LC), was synthesised by coupling stearic anhydride on chitosan, and the surface of LNCs was first modified by this LC by the post-insertion technique. The PEs could be successfully adsorbed on the LNC surface as verified by alternating zeta potential and increase in size. To present a therapeutic application, fondaparinux sodium (FP), a heparin-like synthetic pentasaccharide, was introduced on the LNC surface instead of DS. [ABSTRACT FROM AUTHOR]

Abstract: Over the last decade, Lipid NanoCapsules (LNCs) have been intensively used as effective drug delivery systems; they are classically prepared using a phase-inversion method. Following formulation of the LNCs, the molecular insertion of commercially-available disteraoylphosphatidylethanolamine-peg amphiphiles is performed into the LNC shell, using a post-insertion method, more classically applied with liposomes. The subsequent LNC interfacial modifications are investigated by using size and electrokinetic measurements. More particularly, the length and the nature of the hydrophilic part of the post-inserted surfactant are modified. The results are discussed in order to improve our understanding of post-insertion mechanisms. [Copyright &y& Elsevier]

Abstract: Prolonged vinorelbine exposure is correlated with improved antineoplastic effects, as evidenced by increased response rate in patients receiving continuous infusion. The administration of slow release pegylated liposomal vinorelbine formulation might mimic the pharmacokinetics of a continuous infusion, thus improving antitumor efficacy. But it is hard to prepare pegylated liposome vinorelbine using DSPE-PEG (an extensively used peglipid) because it could induce accelerated drug release. To resolve this problem, “post-insertion” technology was employed to prepare pegylated liposome vinorelbine formulations, which involved the incubation of vinorelbine-containing vesicles with DSPE-PEG micellar solutions. HPLC analysis revealed that after incubation at 60°C for 60min, ∼100% DSPE-PEG could be inserted into the outer monolayer of the vesicles. Moreover, the grafting of peglipid did not induce the release of entrapped vinorelbine irrespective of intraliposomal anions. Drug release experiments indicated that “post-insertion” formulations were more able to retain entrapped drugs than “co-dissolving” formulations. The same phenomenon was observed when both series of formulations were injected in normal mice to compare pharmacokinetic profiles. In L1210 ascitic model, a “post-insertion” formulation with a PEG grafting density of ∼0.5% exhibited the strongest antineoplastic effects, thus it was chosen to be further evaluated in S-180 and RM-1 models, in which the formulation was still more therapeutically active than conventional formulations. In conclusion, using “post-insertion” technology, the potential interaction between DSPE-PEG and vinorelbine could be prevented, thus making it possible to develop pegylated vinorelbine formulations. [Copyright &y& Elsevier]

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]

PEGylated liposomes are one of the useful boron carriers for boron neutron capture therapy (BNCT). Recently, a method of adding PEG after liposome formation (post-insertion) was reported. In this study, we prepared polyborane-encapsulated PEGylated liposomes for BNCT with half the amount of DSPE-PEG of the conventional method using post-insertion technique (post-PEG liposomes), and their usefulness were evaluated in comparison with conventional PEGylated liposomes (pre-PEG liposomes). From the results of physicochemical property measurements, it was confirmed that particle size distributions, surface charge densities, and fixed aqueous layer thicknesses of these liposomes were equivalent. In vitro cytotoxicity and cell uptake tests were also carried out using B16 melanoma and RAW264.7 cells. Polyborane solution and bare liposomes were used for comparison. From the results of these tests, we confirmed that post-PEG liposomes and pre-PEG liposomes have the same influence of PEGylation. To evaluate biodistribution properties at 24 h post-administration, these liposomes and polyborane solution were injected into the tail veins of tumor-bearing mice. Boron concentration and tumor/blood ratios of PEGylated liposomes were 73.2-77.6 µg/g of tumor tissue and 5.5-5.8, respectively. From these results, it was found that by using post-insertion technique, liposomes for BNCT having same effect as the liposome prepared using the conventional method can be prepared with half amount of DSPE-PEG.

Introduction: Liposomes have been extensively investigated as drug delivery vehicles. Immunoliposomes (ILs) are antibody-conjugated liposomes designed to selectively target antigen-expressing cells. ILs can be used to deliver drugs to tumor cells for improving efficacy and reducing toxicity. In addition, ILs can be used in immunoassays, immunotherapy, and imaging. Although there has been extensive coverage on ILs in the literature, only a limited number of clinical trials have been reported and no IL drug has been approved by the FDA., Areas Covered: Factors to consider in developing ILs are discussed, including the choice of antibody or antibody fragment, the formulation of liposomes, and the conjugation chemistry. In addition, challenges and opportunities in clinical development of ILs are discussed. The purpose of this review is to provide an overview on the state of the art of ILs and to discuss potential future developments., Expert Opinion: IL research has had a lengthy history and numerous preclinical studies have yielded encouraging results. However, there are a number of obstacles to clinical translation of ILs. Given the unique capabilities of ILs, its potential for clinical application is underexplored. There is great potential for expanded role for ILs in the clinic and further efforts to this end are warranted., Abbreviations: Ab: antibody; ADCs: antibody-drug conjugates; API: active pharmaceutical ingredient; ADCC: antibody-dependent cellular cytotoxicity; CR: complete remission; cGMP: current good manufacturing practice; DSPE: distearoyl phosphatidylethanolamine; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EPR: enhanced permeability and retention; Fc: fragment crystalline; Tf: transferrin; HACA: human-anti-chimeric antibody; HAHA: human-anti-human antibody; HAMA: human-anti-mouse antibody; HER2: human epidermal growth factor 2; IL: immunoliposome; LNPs: lipid nanoparticles; MRI: magnetic resonance imaging; MTD: maximum tolerated dose; PEG: polyethylene glycol; PET: positron emission tomography; PR: partial response; PSMA: prostate-specific membrane antigen; scFv: single-chain variable fragment; SPECT: single photon emission computed tomography; TTR: transthyretin.

Lactoferrin (Lf) is a potential-targeting ligand for hepatocellular carcinoma (HCC) cells because of its specific binding with asialoglycoprotein receptor (ASGPR). In this present work, a doxorubicin (DOX)-loaded, Lf-modified, polyethylene glycol (PEG)ylated liposome (Lf-PLS) system was developed, and its targeting effect and antitumor efficacy to HCC was also explored. The DOX-loaded Lf-PLS system had spherical or oval vesicles, with mean particle size approximately 100 nm, and had an encapsulation efficiency of 97%. The confocal microscopy and flow cytometry indicated that the cellular uptake of Lf-PLS was significantly higher than that of PEGylated liposome (PLS) in ASGPR-positive cells (P<0.05) but not in ASGPR-negative cells (P>0.05). Cytotoxicity assay by MTT demonstrated that DOX-loaded Lf-PLS showed significantly stronger antiproliferative effects on ASGPR-positive HCC cells than did PLS without the Lf modification (P<0.05). The in vivo antitumor studies on male BALB/c nude mice bearing HepG2 xenografts demonstrated that DOX-loaded Lf-PLS had significantly stronger antitumor efficacy compared with PLS (P<0.05) and free DOX (P<0.05). All these results demonstrated that a DOX-loaded Lf-PLS might have great potential application for HCC-targeting therapy.

Background: Integrins α(v)β(3) and α(v)β(5), both of which specifically recognize the Arg-Gly-Asp (RGD) motif, are overexpressed on many solid tumors and in tumor neovasculature. Thus, coupling the RGD motif to the liposomal surface for achieving active targeting can be a promising strategy for the treatment of tumors., Methods: Cyclo(Arg-Gly-Asp-D-Phe-Cys) (cRGD) was covalently coupled with the liposomal membrane surface, followed by coating with poly(ethylene glycol) (PEG) using the post-insertion technique. The coupling efficiency of cRGD was determined. Doxorubicin as a model anticancer drug was loaded into liposomes using an ammonium sulfate gradient method to investigate the encapsulation efficiency, cellular uptake by the integrin-overexpressing human glioma cell line U87MG in vitro, and pharmacokinetic properties in Sprague-Dawley rats., Results: cRGD was conjugated to the liposomal surface by a thiol-maleimide coupling reaction. The coupling efficiency reached 98%. The encapsulation efficiency of doxorubicin in liposomes was more than 98%. The flow cytometry test result showed that cRGD-modified liposomes (RGD-DXRL-PEG) had higher cell uptake by U87MG cells, compared with nontargeted liposomes (DXRL-PEG). The cellular uptake was significantly inhibited in the presence of excess free cRGD. Both the targeted (t(1/2) = 24.10 hours) and non-targeted (t(1/2) = 25.32 hours) liposomes showed long circulating properties in rat plasma. The area under the curve of the targeted and nontargeted liposomes was 6.4-fold and 8.3-fold higher than that of doxorubicin solution, respectively., Conclusion: This study indicates preferential targeting and long circulating properties for cRGD-modified liposomes in vivo, which could be used as a potential targeted liposomal drug delivery system to treat human glioma.