Post-insertion Technique Research Articles

Improving CEA detection Sensitivity: Carboxyfluorescein-Loaded liposomes in aptamer sandwich assay

This study aimed to develop an effective method for highly sensitive detection of carcinoembryonic antigen (CEA), a tumor biomarker, using an aptasensor sandwich assay based on a liposome loaded with carboxyfluorescein. In this approach, liposomes loaded with carboxyfluorescein (CF) were anchored with a cholesterol-labeled anti-CEA aptamer (Apt1-Lip) using a post-insertion technique. Furthermore, the successful insertion of the aptamer into the liposome outer layer was confirmed through UV absorption, PAGE electrophoresis, dynamic light scattering (DLS), and zeta potential measurements. Additionally, a secondary aptamer was conjugated with magnetic beads via biotin-streptavidin interactions and used as a capture probe (MBs/Apt2). Aptamer-decorated liposomes successfully bound to the CEA antigen in the presence of the target, leading to the formation of a sandwich assay when combined with MBs/Apt2. The specific binding signal was further increased by dissolving the lipid nanospheres with Triton X-100. This liposome-based aptasensor demonstrated a detection limit of 35.48 pg/mL over a wide linear range from 0.1 ng/mL to 100 ng/mL, with high specificity and sensitivity at CEA. Additionally, the proposed approach exhibited notable promise for on-site testing in early clinical diagnosis, as it showed excellent selectivity, satisfactory reproducibility, and good recovery in CEA detection from human serum samples.

Conventional manual technique of post placental IUD insertion versus intra-cesarean post placental introducer withdrawal IUD insertion technique: a new standardized technique for IUD insertion during cesarean section: a randomized controlled trial

BackgroundInserting IUDs during cesarean section reduces the need for more manipulation and discomfort. The current conventional manual technique for IUD insertion during cesarean section is not standardized with many modifications and high rates of expulsion, displacement, missed threads, and discontinuation. This study aims to find a standard technique for IUD insertion during cesarean section with the least possible problems, especially displacement and missed threads.MethodsA randomized controlled study was conducted at Kasr Al-Ainy Maternity Hospital, Cairo University, Egypt. The study was performed over 12 months, from September 2020 to September 2021. Two groups of patients were selected; each group included 420 patients with a desire for IUD insertion during cesarean section. Group (A) (Control group) was subjected to a post-placental intrauterine device (Copper T380) during cesarean section using a conventional manual method; Group (B) (Study group): the IUD (Copper T380) was placed at the top of the uterine fundus using a new technique (intra-cesarean post placental introducer withdrawal IUD insertion technique).ResultsThere was a significant statistical difference between the two groups regarding displacement of the IUDs at the end of puerperium, at 6 months, non-visibility of IUD threads, and continuation of use with p-value < 0.05. There was no significant statistical difference in the term of duration of surgery.ConclusionThe new technique of post-placental IUD insertion can be the standard technique of intra-cesarean section IUD insertion as it is associated with more favorable outcomes among the included women in the form of lower incidence of IUD displacement, non-visibility of IUD threads, and higher rates of continuation without increasing the duration of surgery as compared with the conventional manual technique.Trial registrationClinicalTrial.gov ID: NCT05788354, registration date: 28/03/2023 (retrospectively registered).

Brain-targeted delivery of losartan through functionalized liposomal nanoparticles for management of neurogenic hypertension

There is mounting experimental evidence that blocking angiotensin receptor type 1 activity can prevent the occurrence of hypertension in spontaneously hypertensive rats. Studies have proved this strategy via evasive means, such as intracerebrovascular injections, making clinical translation difficult. This study aimed to develop penetratin and transferrin functionalized liposomes as a delivery tool to safely deliver losartan potassium (an angiotensin receptor blocker) to the brain. Penetratin and transferrin functionalized losartan-loaded liposomes were prepared via the post-insertion technique. Losartan-loaded liposomes were cationic, approximately 150 nm in size, entrapping 66.8 ± 1.5% of losartan. All formulations were well tolerated and internalized by primary and cultured cells in 4 h.Further, the ability to deliver losartan potassium across the blood–brain barrier was evaluated in vivo in Wistar Kyoto rats either in solution or when encapsulated within liposomal nanoparticles. Upon intravenous administration, we did not find a detectable amount of losartan in the brain tissue of rats that received free losartan solution. Contrarily, liposome formulations could deliver losartan to the brain, with a brain AUC and mean resident time of 163.304 ± 13.09 and 8.623 h ± 0.66, respectively. In addition, no toxicity was observed in the animals that received the losartan-loaded liposomes.

Post-insertion technique to introduce targeting moieties in milk exosomes for targeted drug delivery

BackgroundRecently, increased attention has been given on exosomes as ideal nanocarriers of drugs owing to their intrinsic properties that facilitate the transport of biomolecular cargos. However, large-scale exosome production remains a major challenge in the clinical application of exosome-based drug delivery systems. Considering its biocompatibility and stability, bovine milk is a suitable natural source for large-scale and stable exosome production. Because the active-targeting ability of drug carriers is essential to maximize therapeutic efficacy and minimize side effects, precise membrane functionalization strategies are required to enable tissue-specific delivery of milk exosomes with difficulty in post-isolation modification.MethodsIn this study, the membrane functionalization of a milk exosome platform modified using a simple post-insertion method was examined comprehensively. Exosomes were engineered from bovine milk (mExo) with surface-tunable modifications for the delivery of tumor-targeting doxorubicin (Dox). The surface modification of mExo was achieved through the hydrophobic insertion of folate (FA)-conjugated lipids.ResultsWe have confirmed the stable integration of functionalized PE-lipid chains into the mExo membrane through an optimized post-insertion technique, thereby effectively enhancing the surface functionality of mExo. Indeed, the results revealed that FA-modified mExo (mExo-FA) improved cellular uptake in cancer cells via FA receptor (FR)-mediated endocytosis. The designed mExo-FA selectively delivered Dox to FR-positive tumor cells and triggered notable tumor cell death, as confirmed by in vitro and in vivo analyses.ConclusionsThis simple and easy method for post-isolation modification of the exosomal surface may be used to develop milk-exosome-based drug delivery systems.Graphical

Codelivery of π-π Stacked Dual Anticancer Drugs Based on Aloe-Derived Nanovesicles for Breast Cancer Therapy.

To overcome the low efficacy of conventional monotherapeutic approaches that use a single drug, functional nanocarriers loaded with an amalgamation of anticancer drugs have been promising in cancer therapy. Herein, aloe-derived nanovesicles (gADNVs) are modified with an active integrin-targeted peptide (Arg-Gly-Asp, RGD) by the postinsertion technique to deliver indocyanine green (ICG) and doxorubicin (DOX) for efficient breast cancer therapy. We presented for the first time that the π-π stacking interaction can turn the "competitive" relationship of ICG and DOX inside gADNVs into a "cooperative" relationship and enhance their loading efficiency. The dual-drug codelivery nanosystem, denoted as DIARs, was well stable and leakproof, exhibiting high tumor-targeting capability both in vitro and in vivo. Meanwhile, this nanosystem showed significant inhibition of cell growth and migration and induced cell apoptosis with the combination of phototherapy and chemotherapy. Intravenous administration of DIARs exhibited high therapeutic efficacy in a 4T1 tumor-bearing mouse model and exhibited no obvious damage to other organs. Overall, our DIAR nanosystem constitutively integrated the natural and economical gADNVs, π-π stacking interaction based on efficient drug loading, and tumor-targeted RGD modification to achieve an effective combination therapy for breast cancer.

Preparation, characterization, and biodistribution of glutathione PEGylated nanoliposomal doxorubicin for brain drug delivery with a post-insertion approach.

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.

Co-delivery of cisplatin and siRNA through hybrid nanocarrier platform for masking resistance to chemotherapy in lung cancer.

The resistance of cancer cells to chemotherapy has presented a formidable challenge. The current research aims at evaluating whether silencing of the cisplatin efflux promoter gene ABCC3 using siRNA co-loaded with the drug in a nanocarrier improves its efficacy in non-small cell lung cancer (NSCLC). Hybrid nanocarriers (HNCs) comprising lipids and poly(lactic acid-polyethylene glycol) di-block copolymer (PEG-PLA) were prepared for achieving the simultaneous delivery of cisplatin caprylate and ABCC3-siRNA to the cancer cells. PEGylation of the formulated HNCs was carried out using post-insertion technique for imparting long circulation characteristics to the carrier. The optimized formulation exhibited an entrapment efficiency of 71.9±2.2% and 95.83±0.39% for cisplatin caprylate and siRNA respectively. Further, the HNC was found to have hydrodynamic diameter of 153.2±1.76nm and +25.39±0.49mV zeta potential. Morphological evaluation using cryo transmission electron microscopy confirmed the presence of lipid bilayer surrounding the polymeric core in HNCs. The in vitro cellular uptake studies showed improved uptake, while cell viability studies of the co-loaded formulation in A549 cell-line indicated significantly improved cytotoxic potential when compared with drug solution and drug-loaded HNCs; cell cycle analysis indicated increased percentage of cell arrest in G2-M phase compared with drug-loaded HNCs. Further, the gene knock-down study showed that silencing of ABCC3 mRNA might be improved in vitro efficacy of the formulation. The optimized cisplatin and ABCC3 siRNA co-loaded formulation presented significantly increased half-life and tumour regression in A549 xenograft model in BALB/c nude mice. In conclusion, siRNA co-loaded formulation presented reduced drug resistance and increased efficacy, which might be promising for the current cisplatin-based treatments in NSCLC.

Optimization of post-insertion method to conjugate Doxil with anti-CD133 monoclonal antibodies: Investigating the specific binding and cytotoxicity to colorectal cancer cells in vitro

In this paper, Doxil coupled with anti-CD133 monoclonal antibodies made by either routine or optimized post-insertion technique, were compared with respect to their size, drug leakage, release pattern and the number of antibodies conjugated per single liposome. The results demonstrated that the number of antibodies conjugated per liposome in the optimized post-insertion technique was almost two times more than those in the routine post-insertion method. However, the drug release and leakage pattern was almost similar between the two methods. Furthermore, anti-tumor activity and therapeutic efficacy of the preferred CD133–targeted Doxil with Doxil was compared in terms of their in vitro binding, uptake, internalization and cytotoxicity against HT-29 (CD133+) and CHO (CD133-) cells. Flow cytometry analyses and confocal laser scanning microscopy results exhibited a significantly higher cellular uptake, binding and internalization of CD133-targeted Doxil in CD+133 cells relative to Doxil. Cytotoxicity results revealed a lower in vitro inhibitory concentration for CD133–targeted Doxil compared to Doxil. However, CHO (CD133-) cells displayed a similar uptake and in vitro cytotoxicity for both CD133-Doxil and non-targeted Doxil. Therefore, the results of this study can exhibit that specific recognition and binding of antibodies with CD133 receptors on HT-29 cells can result in enhanced cellular uptake, internalization and cytotoxicity. The research suggests further investigation for in vivo studies and may offer proof-of-principle for an active targeting concept.

Poly(Sarcosine) Surface Modification Imparts Stealth-Like Properties to Liposomes.

Circulation lifetime is a crucial parameter for a successful therapy with nanoparticles. Reduction and alteration of opsonization profiles by surface modification of nanoparticles is the main strategy to achieve this objective. In clinical settings, PEGylation is the most relevant strategy to enhance blood circulation, yet it has drawbacks, including hypersensitivity reactions in some patients treated with PEGylated nanoparticles, which fuel the search for alternative strategies. In this work, lipopolysarcosine derivatives (BA-pSar, bisalkyl polysarcosine) with precise chain lengths and low polydispersity indices are synthesized, characterized, and incorporated into the bilayer of preformed liposomes via a post insertion technique. Successful incorporation of BA-pSar can be realized in a clinically relevant liposomal formulation. Furthermore, BA-pSar provides excellent surface charge shielding potential for charged liposomes and renders their surface neutral. Pharmacokinetic investigations in a zebrafish model show enhanced circulation properties and reduction in macrophage recognition, matching the behavior of PEGylated liposomes. Moreover, complement activation, which is a key factor in hypersensitivity reactions caused by PEGylated liposomes, can be reduced by modifying the surface of liposomes with an acetylated BA-pSar derivative. Hence, this study presents an alternative surface modification strategy with similar benefits as the established PEGylation of nanoparticles, but with the potential of reducing its drawbacks.

Polyborane-encapsulated PEGylated Liposomes Prepared Using Post-insertion Technique for Boron Neutron Capture Therapy.

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.

Post-insertion parameters of PEG-derivatives in phosphocholine-liposomes

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-mPEG2000 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-mPEG2000 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 (I2 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.

Synthesis of high functionality and quality mannose-grafted lipids to produce macrophage-targeted liposomes

The mannose receptor, which is responsible for tumor invasion, proliferation, and metastasis in the tumor microenvironment, is overexpressed in tumor-associated macrophages. Mannose is commonly applied to PEGylated liposomes in macrophage-targeted cancer therapy. To develop a high functionality and quality (HFQ) lipid for macrophage-targeted liposomes, we designed a novel mannosylated lipid with improved mannose receptor binding affinity using serine–glycine repeats (SG)n. We synthesized Man(S)-(SG)5-SSK-K(Pal)2 using only a fluorenylmethyloxycarbonyl (Fmoc) protecting group solid-phase peptide synthesis method, which produced a high-quality lipid at a moderately good yield. We then prepared Man-(SG)5/PEGylated liposomes using a post-insertion technique to insert Man(S)-(SG)5-SSK-K(Pal)2 into the PEGylated liposomes. In vitro cell investigations revealed that the Man-(SG)5/PEGylated liposomes effectively associated with mouse peritoneal macrophages by interacting with the mannose receptors. The results suggest that we produced a novel high-quality, highly functional mannosylated lipid that is suitable for clinical drug delivery applications.

Folic acid conjugated nanoliposomes as promising carriers for targeted delivery of bleomycin

Targeted drug delivery has received considerable attention due to its key role in improving therapeutic efficacy and reducing the side effects of anticancer drugs. Bleomycin (BLM) is an anticancer antibiotic with short half-life, low therapeutic and high side effects that limit its clinical applications. This study aims to evaluate the anticancer potential of folate-targeted liposomal bleomycin (FL-BLM) and its free-folate form (L-BLM) on two different cancer cell lines including human cervix carcinoma HeLa, and human breast carcinoma MCF-7 cells. Furthermore, the effect of FL-BLM in induction of apoptosis and cell cycle arrest was studied by flow cytometry. FL-BLM was prepared by thin film hydration method and folic acid was conjugated to nanoliposomes by post insertion technique. Anticancer activity was evaluated by MTT assay. The cytotoxicity of FL-BLM against HeLa cells was significantly increased compared to L-BLM and conventional BLM. Flow cytometry and annexin-V analysis indicated that FL-BLM effectively induced apoptosis and cell-cycle arrest in HeLa cells especially at G2/M phase. In addition, the uptake of FL-BLM by Hela cells was significantly increased as compared to the MCF-7 cells. Overall, our findings indicated that FL-BLM may be promising formulation for targeted drug delivery to folate receptor-positive tumour cells.

Insertion stability of poly(ethylene glycol)-cholesteryl-based lipid anchors in liposome membranes

Liposomes consist of a hydrophilic core surrounded by a phospholipid (PL) bilayer. In human blood, the half-life of such artificial vesicles is limited. To prolong their stability in the circulation, liposomal bilayers can be modified by inserting poly(ethylene glycol) (PEG) molecules using either PL or sterols as membrane anchors. This establishes a hydrophilic steric barrier, reducing the adsorption of serum proteins, recognition and elimination by cells of the immune system. In addition, targeting ligands (such as antibodies) are frequently coupled to the distal end of the PEG chains to direct the vesicles (then called ‘immuno-liposomes’) to specific cell types, such as tumor cells. To our knowledge, experiments on the stability of ligand anchoring have so far only been conducted with PL-based PEGs and not with sterol-based PEGs after insertion via the sterol-based post-insertion technique (SPIT). Therefore, our study examines the insertion stability of PEG-cholesteryl ester (Chol-PEG) molecules with PEG chains of 1000, 1500 and 2000Da molecular mass which have been inserted into the membranes of liposomes using SPIT. For this study we used different acceptor media and multiple analytical techniques, including pulsed-field-gradient nuclear magnetic resonance (PFG-NMR), free-flow electrophoresis, size exclusion chromatography and ultracentrifugation. The obtained data consistently showed that a higher molar mass of PEG chains positively correlates with higher release from the liposome membranes. Furthermore, we could detect and quantify the migration of Chol-PEG molecules from radioactively double-labeled surface-modified liposomes to negatively charged acceptor liposomes via free-flow electrophoresis. Insertion of Chol-PEG molecules into the membrane of preformed liposomes using SPIT is an essential step for the functionalization of liposomes with the aim of specific targeting. For the first time, we present a kinetic analysis of this insertion process using PFG-NMR, showing that insertion into the liposomal membranes takes place within 90s for Chol-PEG1000 molecules.

Dual Drug Delivery Using Lactic Acid Conjugated SLN for Effective Management of Neurocysticercosis.

The debut study was aimed to develop Lactic acid (LA)-conjugated solid lipid nanoparticles (SLN-LA) bearing albendazole (ALB) and prednisolone (PRD) for effective management of neurocysticercosis (NCC). LA was coupled to SLN by post-insertion technique. SLNs were characterized for particle size and size distribution, shape, and percent drug entrapment efficiency. In vitro drug release kinetics, fluorescence study and in vitro transendothelial transport, hematological studies and pharmacokinetic studies were carried out to predict the fullest drug delivery potential. Spherical SLNs (~100 nm) with good drug entrapments (~64 and ~78% for ALB and PRD, respectively) showed in vitro initial fast release (i.e., 20-40% drugs release in 4 h) followed by sustained release for more than 48 h. Fluorescence study and in vitro transendothelial transport depicted selective brain uptake of SLN-LA compared to SLN attributed to carrier mediated transport via monocarboxylic acid transporters (MCT - 1/2/3). Pharmacokinetic parameters such as AUC0-t and AUMC0-t and Cllast showed good drugs withholding capacity of SLNs. Organ distribution studies reflected high accumulation of drugs (ALB, 7.6 ± 0.31%; PRD, 5.21 ± 0.24%) in the brain after 24 h in case of SLN-LA as compared to plain drugs solution. SLN-LA in hematological studies revealed insignificant toxicity to blood cells. The overall study paved the potential advances in brain targeting with synergistic acting drugs for effective management of NCC.

Noninvasive Imaging of Early Venous Thrombosis by 19F Magnetic Resonance Imaging With Targeted Perfluorocarbon Nanoemulsions.

Noninvasive detection of deep venous thrombi and subsequent pulmonary thromboembolism is a serious medical challenge, since both incidences are difficult to identify by conventional ultrasound techniques. Here, we report a novel technique for the sensitive and specific identification of developing thrombi using background-free 19F magnetic resonance imaging, together with α2-antiplasmin peptide (α2AP)-targeted perfluorocarbon nanoemulsions (PFCs) as contrast agent, which is cross-linked to fibrin by active factor XIII. Ligand functionality was ensured by mild coupling conditions using the sterol-based postinsertion technique. Developing thrombi with a diameter<0.8 mm could be visualized unequivocally in the murine inferior vena cava as hot spots in vivo by simultaneous acquisition of anatomic matching 1H and 19F magnetic resonance images at 9.4 T with both excellent signal-to-noise and contrast-to-noise ratios (71±22 and 17±5, respectively). Furthermore, α2AP-PFCs could be successfully applied for the diagnosis of experimentally induced pulmonary thromboembolism. In line with the reported half-life of factor XIIIa, application of α2AP-PFCs>60 minutes after thrombus induction no longer resulted in detectable 19F magnetic resonance imaging signals. Corresponding results were obtained in ex vivo generated human clots. Thus, α2AP-PFCs can visualize freshly developed thrombi that might still be susceptible to pharmacological intervention. Our results demonstrate that 1H/19F magnetic resonance imaging, together with α2AP-PFCs, is a sensitive, noninvasive technique for the diagnosis of acute deep venous thrombi and pulmonary thromboemboli. Furthermore, ligand coupling by the sterol-based postinsertion technique represents a unique platform for the specific targeting of PFCs for in vivo 19F magnetic resonance imaging.

Immuno-magnetoliposomes targeting activated platelets as a potentially human-compatible MRI contrast agent for targeting atherothrombosis

To detect unstable atherosclerotic plaques early and noninvasively would be of great clinical interest. Activated platelets are an interesting molecular target for detecting early lesions or unstable plaques. We therefore developed an MRI contrast agent consisting of magnetoliposomes (ML) linked to an antibody (anti-LIBS) specifically targeting the ligand-induced binding site of the activated GPIIb/IIIa receptor of platelets. ML were prepared by dual centrifugation (DC). ML pegylation up to a total PEG content of 7.5 mol% positively influenced the stability and amount of entrapped SPIOs, and also reduced SPIO-membrane interactions, while higher PEG contents destabilized PEG-ML. Stable anti-LIBS-ML with high amounts of entrapped SPIOs (∼86%, ∼0.22 mol Fe/mol liposomal lipid) and high MRI sensitivity (relaxivity r2 = 422 s−1 mM−1 and r2∗ = 452 s−1 mM−1) were obtained by coupling anti-LIBS to ML in a two-step post-insertion technique. We confirmed specific binding to the GPIIb/IIIa receptor's activated conformation on activated human platelets and cell lines expressing activated GPIIb/IIIa receptor ex vivo. The immuno-ML obtained in this study constitute an important step towards developing a potentially human-compatible MRI contrast agent for the timely detection of plaque rupture by targeting activated platelets.

Apolipoprotein B-100-targeted negatively charged nanoliposomes for the treatment of dyslipidemia

BackgroundAnionic nanoliposomes can interact with serum lipoproteins and regulate lipid metabolism through several mechanisms. This study aimed to evaluate the lipid-modifying effects of anionic immunoliposomes targeted against apoB, an important component of atherogenic lipoproteins. MethodsTwo sets of nanoliposomes (20mM) were prepared with low (including soy phosphatidylcholine [SPC] and egg phosphatidylglycerol [EPG]) and high (including hydrogenated soy phosphatidylcholine [HSPC] and distearoyl phosphatidylglycerol [DSPG]) phase transition temperature values without cholesterol. In each set, the anionic phospholipid (EPG or DSPG) constituted 75% of total phospholipid content. Immunoliposomes were prepared by conjugating a monoclonal antibody against apoB-100 to the liposomal surface using a post-insertion technique. Fluorescently-labeled immunoliposomes were assessed for their uptake by J774.A1 macrophages. Lipid-modifying effects of immunoliposomes were tested at different doses (50, 100 or 200μmole/g weight) using a tyloxapol-induced hyperlipidemic mouse model. Blood sampling was performed 1h after the injection of each immunoliposomal formulation. ResultsApoB-targeted HSPC/DSPG and SPC/EPG nanoliposomes were both taken up by cultured macrophages but the uptake rate was higher with the former formulation. Both immunoliposomal formulations significantly reduced serum LDL-cholesterol concentrations of hyperlipidemic animals at all tested doses (p<0.001) and this effect lasted for at least 48h. Significant reductions of serum levels of apoB, non-HDL-C, total cholesterol and triglycerides, and elevations of HDL-C levels were also observed. ConclusionIntravenous injection of a single dose of apoB-targeted anionic nanoliposomes improves serum lipid profile parameters. These findings might have implications for the treatment of patients with severe dyslipidemias or statin intolerance.

Enhanced pH-Responsiveness, Cellular Trafficking, Cytotoxicity and Long-circulation of PEGylated Liposomes with Post-insertion Technique Using Gemcitabine as a Model Drug

The in vitro and in vivo properties of PEGylated pH-sensitive liposomes (PSL) prepared by pre- and post-insertion techniques were investigated. 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. 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). 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.

Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously

Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification.