PEGylated Liposomal Methyl Prednisolone Succinate does not Induce Infusion Reactions in Patients: A Correlation Between in Vitro Immunological and in Vivo Clinical Studies.

PEGylated nanomedicines are known to induce infusion reactions (IRs) that in some cases can be life-threatening. Herein, we report a case study in which a patient with rare mediastinal and intracardiac IgG4-related sclerosing disease received 8 treatments of intravenously administered PEGylated liposomal methylprednisolone-succinate (NSSL-MPS). Due to the ethical requirements to reduce IRs, the patient received a cocktail of premedication including low dose of steroids, acetaminophen and H2 blockers before each infusion. The treatment was well-tolerated in that IRs, complement activation, anti-PEG antibodies and accelerated blood clearance of the PEGylated drug were not detected. Prior to the clinical study, an in vitro panel of assays utilizing blood of healthy donors was used to determine the potential of a PEGylated drug to activate complement system, elicit pro-inflammatory cytokines, damage erythrocytes and affect various components of the blood coagulation system. The overall findings of the in vitro panel were negative and correlated with the results observed in the clinical phase.

Liposomal steroid nano-drug is superior to steroids as-is in mdx mouse model of Duchenne muscular dystrophy.

Glucocorticosteroids are the most efficacious anti-inflammatory agents and the gold standard treatment in Duchenne muscular dystrophy (DMD). However, their chronic use may lead to severe side effects. We evaluated the use of a novel injectable steroidal nano-drug in mdx mouse model of DMD by comparing the efficacy of nano-liposomes remotely loaded with the steroid prodrug, methylprednisolone hemisuccinate (MPS) with the same steroid as-is, in short (4-weeks) and long-term (58-weeks) treatments. Liposomal-MPS was selectively targeted to the mouse diaphragm, the most dystrophic muscle at early stage of the disease. The bioactivity of the steroidal nano-drug was evidenced by a significant decreased serum TGF-ß and reduced diaphragm macrophage infiltration after short-term treatment. In the long-term, the treatment with liposomal-MPS not only demonstrated improved muscle strength and mobility it also induced lower tibia and lumbar vertebrae osteoporosis indicating much lower bone related adverse effects.

In Vitro Susceptibility of Neisseria gonorrhoeae Strains to Mupirocin, an Antibiotic Reformulated for Parenteral Administration in Nanoliposomes.

Neisseria gonorrhoeae is an urgent antibiotic-resistant threat. This study determined the MICs of mupirocin to be 0.0039 to 0.0625 µg/ml for 94 N. gonorrhoeae strains. Cross-resistance with other antibiotics was not detected. Mupirocin, which is currently limited to topical administration, demonstrated activity by injection when delivered in nanoliposomes. The nanoliposomal formulation of mupirocin is a potential treatment for drug-resistant N. gonorrhoeae.

Understanding the Role of Anti-PEG Antibodies in the Complement Activation by Doxil in Vitro.

Infusion reactions (IRs) are common immune-mediated side effects in patients treated with a variety of drug products, including, but not limited to, nanotechnology formulations. The mechanism of IRs is not fully understood. One of the best studied mechanisms of IRs to nanomedicines is the complement activation. However, it is largely unknown why some patients develop reactions to nanomedicines while others do not, and why some nanoparticles are more reactogenic than others. One of the theories is that the pre-existing anti-polyethylene glycol (PEG) antibodies initiate the complement activation and IRs in patients. In this study, we investigated this hypothesis in the case of PEGylated liposomal doxorubicin (Doxil), which, when used in a clinical setting, is known to induce IRs; referred to as complement activation-related pseudoallergy (CARPA) in sensitive individuals. We conducted the study in vitro using plasma derived from C57BL/6 mice and twenty human donor volunteers. We used mouse plasma to test a library of well-characterized mouse monoclonal antibodies with different specificity and affinity to PEG as it relates to the complement activation by Doxil. We determined the levels of pre-existing polyclonal antibodies that bind to PEG, methoxy-PEG, and PEGylated liposomes in human plasma, and we also assessed complement activation by Doxil and concentrations of complement inhibitory factors H and I in these human plasma specimens. The affinity, specificity, and other characteristics of the human polyclonal antibodies are not known at this time. Our data demonstrate that under in vitro conditions, some anti-PEG antibodies contribute to the complement activation by Doxil. Such contribution, however, needs to be considered in the context of other factors, including, but not limited to, antibody class, type, clonality, epitope specificity, affinity, and titer. In addition, our data contribute to the knowledge base used to understand and improve nanomedicine safety.

Characterization of Pegylated Liposomal Mitomycin C Lipid-Based Prodrug (Promitil) by High Sensitivity Differential Scanning Calorimetry and Cryogenic Transmission Electron Microscopy.

The effect of a lipidated prodrug of mitomycin C (MLP) on the membrane of a pegylated liposome formulation (PL-MLP), also known as Promitil, was characterized through high-sensitivity differential scanning calorimetry (DSC) and cryo-TEM. The thermodynamic analysis demonstrated that MLP led to the formation of heterogeneous domains in the membrane plane of PL-MLP. MLP concentrated in prodrug-rich domains, arranged in high-ordered crystal-like structures, as suggested by the sharp and high enthalpy endotherm in the first heating scanning. After thiolytic cleavage of mitomycin C from MLP by dithiothreitol (DTT) treatment, the crystal-like prodrug domain disappears and a homogeneous membrane with stronger lipid interactions and higher phase transition temperature compared with the blank (MLP-free) liposomes is observed by DSC. In parallel, the rod-like discoid liposomes and the "kissing liposomes" seen by cryo-TEM in the PL-MLP formulation disappear, and liposome mean size and polydispersity increase after DTT treatment. Both MLP and the residual postcleavage lipophilic moiety of the prodrug increased the rigidity of the liposome membrane as indicated by DSC. These results confirm that MLP is inserted in the PL-MLP liposome membrane via its lipophilic anchor, and its mitomycin C moiety located mainly at the region of the phospholipid glycerol backbone and polar headgroup. We hypothesize that π-π stacking between the planar aromatic rings of the mitomycin C moieties leads to the formation of prodrug-rich domains with highly ordered structure on the PL-MLP liposome membrane. This thermodynamically stable conformation may explain the high stability of the PL-MLP formulation. These results also provide us with an interesting example of the application of high sensitivity DSC in understanding the composition-structure-behavior dynamics of liposomal nanocarriers having a lipid-based drug as pharmaceutical ingredient.

Anticoagulants Influence the Performance of In Vitro Assays Intended for Characterization of Nanotechnology-Based Formulations.

The preclinical safety assessment of novel nanotechnology-based drug products frequently relies on in vitro assays, especially during the early stages of product development, due to the limited quantities of nanomaterials available for such studies. The majority of immunological tests require donor blood. To enable such tests one has to prevent the blood from coagulating, which is usually achieved by the addition of an anticoagulant into blood collection tubes. Heparin, ethylene diamine tetraacetic acid (EDTA), and citrate are the most commonly used anticoagulants. Novel anticoagulants such as hirudin are also available but are not broadly used. Despite the notion that certain anticoagulants may influence assay performance, a systematic comparison between traditional and novel anticoagulants in the in vitro assays intended for immunological characterization of nanotechnology-based formulations is currently not available. We compared hirudin-anticoagulated blood with its traditional counterparts in the standardized immunological assay cascade, and found that the type of anticoagulant did not influence the performance of the hemolysis assay. However, hirudin was more optimal for the complement activation and leukocyte proliferation assays, while traditional anticoagulants citrate and heparin were more appropriate for the coagulation and cytokine secretion assays. The results also suggest that traditional immunological controls such as lipopolysaccharide (LPS ) are not reliable for understanding the role of anticoagulant in the assay performance. We observed differences in the test results between hirudin and traditional anticoagulant-prepared blood for nanomaterials at the time when no such effects were seen with traditional controls. It is, therefore, important to recognize the advantages and limitations of each anticoagulant and consider individual nanoparticles on a case-by-case basis.

Celecoxib Encapsulation in ß-Casein Micelles: Structure, Interactions, and Conformation.

ß-Casein is a 24 kDa natural protein that has an open conformation and almost no folded or secondary structure, and thus is classified as an intrinsically unstructured protein. At neutral pH, ß-casein has an amphiphilic character. Therefore, in contrast to most unstructured proteins that remain monomeric in solution, ß-casein self-assembles into well-defined core-shell micelles. We recently developed these micelles as potential carriers for oral administration of poorly water-soluble pharmaceuticals, using celecoxib as a model drug. Herein we present deep and precise insight into the physicochemical characteristics of the protein-drug formulation, both in bulk solution and in dry form, emphasizing drug conformation, packing properties and aggregation state. In addition, the formulation is extensively studied in terms of structure and morphology, protein/drug interactions and physical stability. Particularly, NMR measurements indicated strong drug-protein interactions and noncrystalline drug conformation, which is expected to improve drug solubility and bioavailability. Small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) were combined for nanostructural characterization, proving that drug-protein interactions lead to well-defined spheroidal micelles that become puffier and denser upon drug loading. Dynamice light scattering (DLS), turbidity measurements, and visual observations complemented the analysis for determining formulation structure, interactions, and stability. Additionally, it was shown that the loaded micelles retain their properties through freeze-drying and rehydration, providing long-term physical and chemical stability. Altogether, the formulation seems greatly promising for oral drug delivery.

In vitro experiments showing enhanced release of doxorubicin from Doxil® in the presence of ammonia may explain drug release at tumor site.

The anticancer nanodrug Doxil®, a pegylated liposomal doxorubicin (PLD), accumulates at the tumor site due to the enhanced permeability and retention effect. However, the mechanism of doxorubicin release from the liposome within the tumor is unknown. We propose that ammonia produced at the tumor site by glutaminolysis enhances release. Using tumor cells in culture, we show that PLD, when ammonia is present, kills tumor cells with an efficacy similar to that of free doxorubicin, while PLD without ammonia and ammonia without PLD have very poor cytotoxicity. We confirm in tumor mouse models that ammonium/ammonia levels measured at the tumors are in the millimolar range, much higher than in the plasma of these mice. This is a new concept of stimulus-response, therapeutically efficacious drug release in tumors, with ammonia derived from tumor cell glutaminolysis acting as the stimulus. There may also be additional microenvironment-related variables that influence therapeutic efficacy. FROM THE CLINICAL EDITOR: The use of liposomal platform as a drug carrier has brought success to Doxil. Nonetheless, the underlying mechanism of drug release at tumor site and subsequent tumor killing was largely unknown. In this article, the authors demonstrated in their experiments that higher ammonia level in the tumor environment was the main mechanism for drug release.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA-Lipid Complexes.

Despite the worldwide success of mRNA-LNP Covid-19 vaccines, the nanoscale structures of these formulations are still poorly understood. To fill this gap, we used a combination of atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and the determination of the intra-LNP pH gradient to analyze the nanoparticles (NPs) in BNT162b2 (Comirnaty), comparing it with the well-characterized PEGylated liposomal doxorubicin (Doxil). Comirnaty NPs had similar size and envelope lipid composition to Doxil; however, unlike Doxil liposomes, wherein the stable ammonium and pH gradient enables accumulation of 14C-methylamine in the intraliposomal aqueous phase, Comirnaty LNPs lack such pH gradient in spite of the fact that the pH 4, at which LNPs are prepared, is raised to pH 7.2 after loading of the mRNA. Mechanical manipulation of Comirnaty NPs with AFM revealed soft, compliant structures. The sawtooth-like force transitions seen during cantilever retraction imply that molecular strands, corresponding to mRNA, can be pulled out of NPs, and the process is accompanied by stepwise rupture of mRNA-lipid bonds. Unlike Doxil, cryo-TEM of Comirnaty NPs revealed a granular, solid core enclosed by mono- and bilipid layers. Negative staining TEM shows 2-5 nm electron-dense spots in the LNP's interior that are aligned into strings, semicircles, or labyrinth-like networks, which may imply cross-link-stabilized RNA fragments. The neutral intra-LNP core questions the dominance of ionic interactions holding together this scaffold, raising the possibility of hydrogen bonding between mRNA and the lipids. Such interaction, described previously for another mRNA/lipid complex, is consistent with the steric structure of the ionizable lipid in Comirnaty, ALC-0315, displaying free ═O and -OH groups. It is hypothesized that the latter groups can get into steric positions that enable hydrogen bonding with the nitrogenous bases in the mRNA. These structural features of mRNA-LNP may be important for the vaccine's activities in vivo.

Anti-PEG antibodies before and after a first dose of Comirnaty® (mRNA-LNP-based SARS-CoV-2 vaccine).

The early and massive vaccination campaign in Israel with the mRNA-LNP Comirnaty® (Pfizer-BioNTech) vaccine against the SARS-CoV-2 virus made available large amounts of data regarding the efficacy and safety of this vaccine. Adverse reactions to mRNA-based SARS-CoV-2 vaccines are rare events, but due to large mediatic coverage they became feared and acted as a potential source of delay for the vaccination of the Israeli population. The experience with the reactogenicity of the polyethylene glycol (PEG) moiety of PEGylated liposomes, PEGylated proteins and other PEGylated drugs raised the fear that similar adverse effects can be associated with the PEG lipid which is an essential component of currently used mRNA-LNP vaccines against COVID-19. In this study we quantified the levels of anti-PEG IgG, IgM and IgE present in the blood of 79 volunteers immediately before and 3 weeks after receiving a first dose of Comirnaty® vaccine. Our in vitro results show that different humanized anti-PEG antibodies bind the PEGylated nano-liposomes in a concentration-dependent manner, but they bind with a lower affinity to the Comirnaty vaccine, despite it having a high mole% of neutral PEG2000-lipid on its surface. We found an increase in IgG concentration in the blood 3 weeks after the first vaccine administration, but no increase in IgM or IgE. In addition, no severe signs of adverse reactions to the Comirnaty vaccine were observed in the population studied despite the significant pre-existing high titers of IgG before the first dose of vaccine in 2 donors.

Resolvin D1 improves allograft osteointegration and directly enhances osteoblasts differentiation.

Introduction: Allografts are the most common bone grafts for repairing osseous defects. However, their use is associated with an increased risk for infections, donor disease transmission and osteointegration deficiency. Resolvin D1 (RvD1) is an endogenous lipid with a scientifically proven pivotal role in inflammation resolution and osteoclastogenesis inhibition. Yet, its biological relevance as a potential bone regenerative drug has been scarcely studied. Here, we aim to investigate the RvD1 effect on allograft osteointegration in the alveolar bone regeneration (ABR) murine model. Methods: ABR model consisted of osseous defects that were generated by the extraction of the maxillary first molar in C57BL/6 mice. The sockets were filled with allograft and analyzed via RNA sequencing. Then they were locally injected with either RvD1 or saline via single or repeated administrations. The mice were sacrificed 2W after the procedure, and regenerated sites were analyzed using µCT and histology. First, MC3T3-E1 preosteoblasts were plated with IL-17 pro-inflammatory medium, and RANKL/OPG ratio was measured. Secondly, the MC3T3-E1 were cultured w/o RvD1, for 3W. Osteoblasts' markers were evaluated in different days, using qRT-PCR and Alizarin Red staining for calcified matrix. Results: In vivo, neither allograft alone nor single RvD1 administration promote bone regeneration in comparison to the control of spontaneous healing and even triggered an elevation in NR1D1 and IL1RL1 expression, markers associated with inflammation and inhibition of bone cell differentiation. However, repeated RvD1 treatment increased bone content by 135.92% ± 45.98% compared to its specific control, repeated sham, and by 39.12% ± 26.3% when compared to the spontaneous healing control group (n=7/group). Histologically, repeated RvD1 reduced the number of TRAP-positive cells, and enhanced allograft osteointegration with new bone formation. In vitro, RvD1 rescued OPG expression and decreased RANKL/OPG ratio in IL-17 pro-inflammatory conditions. Furthermore, RvD1 increased the expression of RUNX2, OSX, BSP and OC/BGLAP2 and the mineralized extracellular matrix during MC3T3-E1 osteoblasts differentiation. Conclusions: Repeated administrations of RvD1 promote bone regeneration via a dual mechanism: directly, via enhancement of osteoblasts' differentiation and indirectly, through reduction of osteoclastogenesis and RANKL/OPG ratio. This suggests that RvD1 may be a potential therapeutic bioagent for osseous regeneration following allograft implantation.

Therapeutic efficacy and pharmacokinetics of liposomal-cannabidiol injection: a pilot clinical study in dogs with naturally-occurring osteoarthritis.

Introduction: Osteoarthritis is a common disease in dogs resulting in chronic pain and decreased wellbeing. Common analgesics such as non-steroidal anti-inflammatories may fail to control pain and can produce major adverse effects. Study objectives were to evaluate pharmacokinetics, therapeutic efficacy, and safety of subcutaneous liposomal-cannabidiol (CBD) as an additional analgesic therapy in dogs suffering from naturally-occurring osteoarthritis. Methods: Six such dogs were recruited following ethics approval and owner consent. Dogs were administered a single subcutaneous injection of 5 mg/kg liposomal-CBD. Plasma concentrations of CBD, blood work, activity monitoring collar data, wellbeing questionnaire (owners) and pain scoring (veterinarian) were performed at baseline and monitored up to six weeks following intervention. Data overtime were compared with baseline using linear-regression mixed-effects. P-value was set at 0.05. Results: CBD plasma concentrations were observed for 6 weeks; median (range) peak plasma concentration (Cmax) was 45.2 (17.8-72.5) ng/mL, time to Cmax was 4 (2-14) days and half-life was 12.4 (7.7-42.6) days. Median (range) collar activity score was significantly increased on weeks 5-6; from 29 (17-34) to 34 (21-38). Scores of wellbeing and pain evaluations were significantly improved at 2-3 weeks; from 69 (52-78) to 53.5 (41-68), and from 7.5 (6-8) to 5.5 (5-7), respectively. The main adverse effect was minor local swelling for several days in 5/6 dogs. Conclusion: Liposomal-CBD administered subcutaneously produced detectable CBD plasma concentrations for 6 weeks with minimal side effects and demonstrated reduced pain and increased wellbeing as part of multimodal pain management in dogs suffering from osteoarthritis. Further placebo-controlled studies are of interest.

Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome.

Hypersensitivity reactions to liposomal drugs, often observed with Doxil and AmBisome, can arise from activation of the complement (C) system by phospholipid bilayers. To understand the mechanism of this adverse immune reaction called C activation-related pseudoallergy (CARPA), we analyzed the relationship among liposome features, C activation in human serum in vitro, and liposome-induced cardiovascular distress in pigs, a model for human CARPA. Among the structural variables (surface charge, presence of saturated, unsaturated, and PEGylated phospholipids, and cisplatin vs. doxorubicin inside liposomes), high negative surface charge and the presence of doxorubicin were significant contributors to reactogenicity both in vitro and in vivo. Morphological analysis suggested that the effect of doxorubicin might be indirect, via distorting the sphericity of liposomes and, if leaked, causing aggregation. The parallelism among C activation, cardiopulmonary reactions in pigs, and high rate of hypersensitivity reactions to Doxil and AmBisome in humans strengthens the utility of the applied tests in predicting the risk of CARPA. FROM THE CLINICAL EDITOR: The authors studied complement activation-related pseudoallergy (CARPA) in a porcine model and demonstrate that high negative surface charge and drug effects leading to distortion of liposome sphericity might be the most critical factors leading to CARPA. The applied tests might be used to predict CARPA in humans.

A Case Report of Subcutaneously Injected Liposomal Cannabidiol Formulation Used as a Compassion Therapy for Pain Management in a Dog.

A 14-year-old intact mixed breed dog (26 kg) was submitted for a novel cannabidiol (CBD) analgesic treatment. The dog was cachectic and had a testicular neoplasia, hip and elbow osteoarthritis and severe cervical pain. Analgesic treatment included canine osteoarthritic supplement, robencoxib and gabapentin. An additional liposomal CBD injectable formulation at 5 mg/kg was administered subcutaneously between the shoulder blades. The dog was monitored using an activity monitoring collar (PetPace), owner wellbeing questionnaire (Canine Brief Pain Inventory; CBPI), pain interactive visual analog scale (iVAS), blood work and CBD plasma concentrations. A week from the injection and up to 3 weeks afterwards the dog had improved CBPI and iVAS pain scores, and increased collar activity scores. CBD was quantified in plasma for 28 days. Due to disease progression, further difficulty to rise and walk, and relapse to pain after 3 weeks, the owners requested a second liposomal CBD injection, which was performed 4 weeks following the first injection using 3 mg/kg dose. Two days later, the dog was found dead in the yard under direct sun, while environmental temperature was 37°C. Major findings on necropsy revealed evidence of heat stroke and severe cervical disc protrusion with spinal hematoma, none related to liposomal CBD. In conclusion, subcutaneous liposomal CBD produced quantifiable CBD plasma concentrations for 28 days and may be an effective additional treatment as part of multimodal pain management in dogs.

Applying lessons learned from nanomedicines to understand rare hypersensitivity reactions to mRNA-based SARS-CoV-2 vaccines.

After over a billion of vaccinations with messenger RNA-lipid nanoparticle (mRNA-LNP) based SARS-CoV-2 vaccines, anaphylaxis and other manifestations of hypersensitivity can be considered as very rare adverse events. Although current recommendations include avoiding a second dose in those with first-dose anaphylaxis, the underlying mechanisms are unknown; therefore, the risk of a future reaction cannot be predicted. Given how important new mRNA constructs will be to address the emergence of new viral variants and viruses, there is an urgent need for clinical approaches that would allow a safe repeated immunization of high-risk individuals and for reliable predictive tools of adverse reactions to mRNA vaccines. In many aspects, anaphylaxis symptoms experienced by the affected vaccine recipients resemble those of infusion reactions to nanomedicines. Here we share lessons learned over a decade of nanomedicine research and discuss the current knowledge about several factors that individually or collectively contribute to infusion reactions to nanomedicines. We aim to use this knowledge to inform the SARS-CoV-2 lipid-nanoparticle-based mRNA vaccine field.

Resolvin D1 shows osseous-protection via RANK reduction on monocytes during orthodontic tooth movement.

The study aimed to investigate the role of RvD1 in acute and prolonged sterile inflammation and bone remodeling. A mouse model of sterile inflammation that involves bone resorption was used to examine endogenous RvD1 kinetics during inflammation. Application of exogenous RvD1 significantly inhibited bone remodeling via osteoclast reduction, alongside an anti-inflammatory secretome shift, increased macrophages recruitment and reduction of T-cytotoxic cells. In vitro and in vivo, RvD1 led to significant reduction in RANK expression which reduce osteoclastogenesis in a dose-dependent manner. Taken together, the data shows a dual role for RvD1, as a potent immunoresolvent agent alongside an osteoresolvent role, showing a potential therapeutic agent in bone resorption associated inflammatory conditions.

Interactions between adsorbed hydrogenated soy phosphatidylcholine (HSPC) vesicles at physiologically high pressures and salt concentrations.

Using a surface force balance, we measured normal and shear interactions as a function of surface separation between layers of hydrogenated soy phosphatidylcholine (HSPC) small unilamellar vesicles (SUVs) adsorbed from dispersion at physiologically high salt concentrations (0.15 M NaNO3). Cryo-scanning electron microscopy shows that each surface is coated by a close-packed HSPC-SUV layer with an overlayer of liposomes on top. A clear attractive interaction between the liposome layers is seen upon approach and separation, followed by a steric repulsion upon further compression. The shear forces reveal low friction coefficients (µ = 0.008-0.0006) up to contact pressures of at least 6 MPa, comparable to those observed in the major joints. The spread in µ-values may be qualitatively accounted for by different local liposome structure at different contact points, suggesting that the intrinsic friction of the HSPC-SUV layers at this salt concentration is closer to the lower limit (µ = ~0.0006). This low friction is attributed to the hydration lubrication mechanism arising from rubbing of the hydrated phosphocholine-headgroup layers exposed at the outer surface of each liposome, and provides support for the conjecture that phospholipids may play a significant role in biological lubrication.

Liposomal Bupivacaine (Bupigel) Demonstrates Minimal Local Nerve Toxicity in a Rabbit Functional Model.

We previously reported the development of a novel formulation of an ultra-long-acting local anesthetic based on bupivacaine encapsulated in large multivesicular liposomes (Bupisomes) embedded in hydrogel. This formulation (Bupigel) prolonged bupivacaine release from the formulation in dissolution-like studies in vitro and analgesia in vivo in mouse, rat, and pig models. In this study we assessed Bupigel neurotoxicity on rabbit sciatic nerve using histopathology and electrophysiologic testing. Sciatic nerves of both hind limbs were injected dropwise with different formulations. Nerve conduction studies and needle electromyography two weeks after perineural administration showed signs of neural damage after injection of free lidocaine and bupivacaine, while there was no sign of neural damage after injection with saline, demonstrating the validity of the method. This test also did not show evidence of motor or sensory nerve damage after injection with liposomal bupivacaine at a dose 10-times higher than free bupivacaine. Histologically, signs of neural damage could be observed with lidocaine. Nerves injected with Bupigel showed mild signs of inflammation and small residues of hydrogel in granulomas, indicating a long residence time of the hydrogel at the site of injection, but no histopathological signs of nerve damage. This demonstrated that early signs of neural damage were detected electrophysiologically, showing the usefulness and sensitivity of electrodiagnostic testing in detection of neural damage from new formulations.

Effect of the ammonium salt anion on the structure of doxorubicin complex and PEGylated liposomal doxorubicin nanodrugs.

BACKGROUND: In Doxil®, PEGylated nanoliposomes are created by hydration of the lipids in ammonium sulfate, and are remotely loaded with doxorubicin by a transmembrane ammonium gradient. The ammonium sulfate is then removed from the external aqueous phase, surrounding the liposomes, and replaced by an isoosmotic sucrose solution in 10 mM histidine buffer at pH 6.5. METHODS: We prepared PEGylated liposomal doxorubicin (PLD) with a series of ammonium monovalent salts that after remote loading became the intraliposome doxorubicin counteranions. We analyzed the liposomes by solution X-ray scattering, differential scanning calorimetry, and electron micropscopy. RESULTS: PLDs prepared with sulfonic acid derivatives as counteranion exhibited chemical and physical stabilities. We determined the effect of these ammonium salt counteranions on the structure, morphology, and thermotropic behavior of the PEGylated nanoliposomes, formed before and after doxorubicin loading, and the bulk properties of the doxorubicin-counteranion complexes. By comparing the structure of the doxorubicin complexes in the bulk and inside the nanoliposomes, we revealed the effect of confinement on the structure and doxorubicin release rate for each of the derivatives of the ammonium sulfonic acid counteranions. CONCLUSIONS: We found that the extent and direction of the doxorubicin confinement effect and its release rate were strongly dependent on the type of counteranion. The counteranions, however, neither affected the structure and thermotropic behavior of the liposome membrane, nor the thickness and density of the liposome PEG layers. In an additional study, it was demonstrated that PLD made with ammonium-methane sulfonate exhibit a much lower Hand and Foot syndrome. GENERAL SIGNIFICANCE: The structure, physical state, and pharmacokinetics of doxorubicin in PEGylated nanoliposomes, prepared by transmembrane remote loading using gradients of ammonium salts, strongly depend on the counteranions.