Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.     

Quaternary dynamics and plasticity underlie small heat shock protein chaperone function

Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that prevent protein aggregation by binding clients destabilized during cellular stress. Here we probe the architecture and dynamics of complexes formed between an oligomeric sHSP and client by employing unique mass spectrometry strategies. We observe over 300 different stoichiometries of interaction, demonstrating that an ensemble of structures underlies the protection these chaperones confer to unfolding clients. This astonishing heterogeneity not only makes the system quite distinct in behavior to ATP-dependent chaperones, but also renders it intractable by conventional structural biology approaches. We find that thermally regulated quaternary dynamics of the sHSP establish and maintain the plasticity of the system. This extends the paradigm that intrinsic dynamics are crucial to protein function to include equilibrium fluctuations in quaternary structure, and suggests they are integral to the sHSPs’ role in the cellular protein homeostasis network.     

Lineage-related and particle size-dependent cytotoxicity of chitosan nanoparticles on mouse bone marrow-derived hematopoietic stem and progenitor cells

Chitosan nanoparticles (CSNPs) have potential applications in stem cell research. In this study, ex vivo cytotoxicity of CSNPs on mouse bone marrow-derived (MBMCs) hematopoietic stem and progenitor cells (HSPCs) was determined. MBMCs were exposed to CSNPs of different particle sizes at various concentrations for up to 72 h. Cytotoxicity effect of CSNPs on MBMCs was determined using MTT, Live/Dead Viability/Cytotoxicity assays and flow cytometry analysis of surface antigens on HSCs (Sca-1⁺), myeloid-committed progenitors (CD11b⁺, Gr-1⁺), and lymphoid-committed progenitors (CD45⁺, CD3e⁺). At 24 h incubation, MBMCs' viability was not affected by CSNPs. At 48 and 72 h, significant reduction was detected at higher CSNPs concentrations. Small CSNPs (200 nm) significantly reduced MBMCs' viability while medium-sized particle (∼400 nm) selectively promoted MBMCs growth. Surface antigen assessment demonstrated lineage-dependent effect. Significant decrease in Sca-1⁺ cells percentage was observed for medium-sized particle at the lowest CSNPs concentration. Meanwhile, reduction of CD11b⁺ and Gr-1⁺ cells percentage was detected at high and intermediate concentrations of medium-sized and large CSNPs. Percentage of CD45⁺ and CD3e⁺ cells along with ROS levels were not significantly affected by CSNPs. In conclusion, medium-sized and large CSNPs were relatively non-toxic at lower concentrations. However, further investigations are necessary for therapeutic applications.     

Dibucaine in Ionic-Gradient Liposomes: Biophysical,Toxicological, and Activity Characterization

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 μM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.     

Nanoformulations of anticancer FGFR inhibitors with improved therapeutic index

Fibroblast growth factor receptor (FGFR) inhibitors like ponatinib and nintedanib are clinically approved for defined cancer patient cohorts but often exert dose-limiting adverse effects. Hence, we encapsulated the FGFR inhibitors ponatinib, PD173074, and nintedanib into polylactic acid nanoparticles and liposomes to enable increased tumor accumulation/specificity and reduce side effects. Different methods of drug loading were tested and the resulting formulations compared regarding average size distribution as well as encapsulation efficiency. Appropriate encapsulation levels were achieved for liposomal preparations only. Nanoencapsulation resulted in significantly decelerated uptake kinetics in vitro with clearly decreased short-term (up to 72?h) cytotoxicity at higher concentrations. However, in long-term clonogenic assays liposomal formations were equally or even more active as compared to the free drugs. Accordingly, in an FGFR inhibitor-sensitive murine osteosarcoma transplantation model (K7M2), only liposomal but not free ponatinib resulted in significant tumor growth inhibition (by 60.4%) at markedly reduced side effects.     

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as “accelerated blood clearance (ABC) phenomenon.” Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose–dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.     

Codelivery of Adriamycin and P-gp Inhibitor Quercetin Using PEGylated Liposomes to Overcome Cancer Drug Resistance

Transmembrane protein P-gp's overexpression at the drug-resistant cell membrane is the most important characteristic of multidrug resistance (MDR). Quercetin (QUE) can effectively suppress the function of P-gp to reverse MDR. This study uses QUE as the P-gp inhibitor andfilm-ultrasound technique with ammonium sulfate transmembrane gradient method to prepare long-circulating liposomes simultaneously encapsulating QUE and Adriamycin (doxorubicin) (AMD/DOX). The optimal conditions for the preparation of AMD_QUE_long-circulating liposomes (SLs) are as follows: hydrogenated soybean phospholipids (HSPC):cholesterol:DSPE-PEG 2000 = 73.07:24.36:2.57 mol/mol, QUE:HSPC = 1:20 mol/mol, AMD:HSPC = 1:7.9 w/w (NH₄₎₂SO₄ 0.15 mol/L, drug loaded (AMD) at 55°C for 25 min). The average encapsulation efficiency of AMD and QUE was 97.49% and 95.50%, respectively. The average particle size is 85 nm (n = 3), and the average zeta potential is ?14.9 mV. First, the pharmacokinetic study proved that codelivery liposomes enveloping QUE and AMD (AMD_QUE_SL) can obviously increase the blood concentration of AMD (C_max: 140.50 ± 32.37 μg/mL) and extend the half-life period of AMD in plasma (t_1/2:14.02 ± 1.54 h). Second, AMD_QUE_SL can obviously enhance the cell toxicity to AMD-resistant cell strains (HL-6/ADR and MCF-7/ADR), and the reverse effects on the resistance of HL-6/ADR and MCF-7/ADR is increased to 4.81-fold and 3.21-fold, respectively. Third, according to the in vivo pharmacodynamic study, the relative tumor volume and relative tumor growth of the AMD_QUE_SL group were the lowest. The inhibition rate of tumor growth of this group was the highest. It can be concluded that AMD_QUE_SL can effectively reverse MDR, lower cardiac toxicity of AMD in clinical treatment, and improve the clinical treatment effect of AMD.     

The Hypotonic Environmental Changes Affect Liposomal Formulations for Nose-to-Brain Targeted Drug Delivery

Systemic administration of drugs is ineffective in the treatment of central nervous system disorders because of the blood-brain barrier. Nasal administration has been suggested as an alternative administration route as drugs absorbed in the olfactory epithelium bypass the blood-brain barrier and reach the brain within minutes. However, the nasal mucosa properties (e.g., tonicity, pH) are not constant because of physiological and environmental factors, and this might limit the therapeutic outcome of nanocarrier-based formulations. To shine light on the impact of environmental ionic strength on nanocarrier-based formulations, we have studied how liposomal formulations respond to the change of tonicity of the external environment. Large unilamellar vesicles loaded with 6 different drugs were exposed to different hypotonic environments, creating an osmotic gradient within the inner core and external environment of the liposomes up to 650 mOsm/kg. Both size and polydispersity of liposomes were significantly affected by tonicity changes. Moreover, the release kinetics of hydrophilic and lipophilic drugs were largely enhanced by hypotonic environments. These results clearly demonstrate that the environmental ionic strength has an impact on liposomal formulation stability and drug release kinetics and it should be considered when liposomal formulations for nose-to-brain targeted drug delivery are designed.     

A Nonionic Polyethylene Oxide (PEO) Surfactant Model: Experimental and Molecular Dynamics Studies of Kolliphor EL

Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6_DBW and 2016H66 force field parameters for polyethoxylate chains. To compare the computational models to experimental measurements, we investigated the phase behavior of KOL using nephelometry, dynamic light scattering, cross-polarized microscopy, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The potential for digestion of KOL was also evaluated using an in vitro digestion experiment. We found that the size and spherical morphology of the KOL colloids at low concentrations was reproduced by the MD models as well as the growing interactions between the aggregates to from rod-like structures at high concentrations. We believe that this model reproduces the phase behavior of KOL relevant to drug absorption and that it can be used in whole formulation simulations to accelerate the formulation development.