Cerebral malaria is the most severe complication of Plasmodium falciparum infection, and a leading cause of death in children under the age of five in malaria-endemic areas. We report high therapeutic efficacy of a novel formulation of liposome-encapsulated water-soluble glucocorticoid prodrugs, and in particular β-methasone hemisuccinate (BMS), for treatment of experimental cerebral malaria (ECM), using the murine P. berghei ANKA model. BMS is a novel derivative of the potent steroid β-methasone, and was specially synthesized to enable remote loading into nano-sterically stabilized liposomes (nSSL), to form nSSL-BMS. The novel nano-drug, composed of nSSL remote loaded with BMS, dramatically improves drug efficacy and abolishes the high toxicity seen upon administration of free BMS. nSSL-BMS reduces ECM rates in a dose-dependent manner and creates a survival time-window, enabling administration of an antiplasmodial drug, such as artemisone. Administration of artemisone after treatment with the nSSL-BMS results in complete cure. Treatment with BMS leads to lower levels of cerebral inflammation, demonstrated by changes in cytokines, chemokines, and cell markers, as well as diminished hemorrhage and edema, correlating with reduced clinical score. Administration of the liposomal formulation results in accumulation of BMS in the brains of sick mice but not of healthy mice. This steroidal nano-drug effectively eliminates the adverse effects of the cerebral syndrome even when the treatment is started at late stages of disease, in which disruption of the blood-brain barrier has occurred and mice show clear signs of neurological impairment. Overall, sequential treatment with nSSL-BMS and artemisone may be an efficacious and well-tolerated therapy for prevention of CM, elimination of parasites, and prevention of long-term cognitive damage.

Doxil®, the first FDA-approved nano-drug (1995), is based on three unrelated principles: (i) prolonged drug circulation time and avoidance of the RES due to the use of PEGylated nano-liposomes; (ii) high and stable remote loading of doxorubicin driven by a transmembrane ammonium sulfate gradient, which also allows for drug release at the tumor; and (iii) having the liposome lipid bilayer in a "liquid ordered" phase composed of the high-T(m) (53 °C) phosphatidylcholine, and cholesterol. Due to the EPR effect, Doxil is "passively targeted" to tumors and its doxorubicin is released and becomes available to tumor cells by as yet unknown means. This review summarizes historical and scientific perspectives of Doxil development and lessons learned from its development and 20 years of its use. It demonstrates the obligatory need for applying an understanding of the cross talk between physicochemical, nano-technological, and biological principles. However, in spite of the large reward, ~2 years after Doxil-related patents expired, there is still no FDA-approved generic "Doxil" available.

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.

Orthodontic tooth movement (OTM) is a "sterile" inflammatory process. The present study aimed to reveal the underlying biological mechanisms, by studying the force associated-gene expression changes, in a time-dependent manner. Ni-Ti springs were set to move the upper 1st-molar in C57BL/6 mice. OTM was measured by μCT. Total-RNA was extracted from tissue blocks at 1,3,7 and 14-days post force application, and from two control groups: naïve and inactivated spring. Gene-expression profiles were generated by next-generation-RNA-sequencing. Gene Set Enrichment Analysis, K-means algorithm and Ingenuity pathway analysis were used for data interpretation. Genes of interest were validated with qRT-PCR. A total of 3075 differentially expressed genes (DEGs) were identified, with the greatest number at day 3. Two distinct clusters patterns were recognized: those in which DEGs peaked in the first days and declined thereafter (tissue degradation, phagocytosis, leukocyte extravasation, innate and adaptive immune system responses), and those in which DEGs were initially down-regulated and increased at day 14 (cell proliferation and migration, cytoskeletal rearrangement, tissue homeostasis, angiogenesis). The uncovering of novel innate and adaptive immune processes in OTM led us to propose a new term "Immunorthodontics". This genomic data can serve as a platform for OTM modulation future approaches.

It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions.

Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with adverse effects related to drug distribution into non-diseased tissues, a situation which attracts a rational design of a targeted treatment confined to the inflamed mucosa. Upon activation of immune cells, transferrin receptor (TfR) expression increases at their surface. Because TfR is expressed in all cell types we hypothesized that its cell surface levels are regulated also in enterocytes. We, therefore, compared TfR expression in healthy and inflamed human colonic mucosa, as well as healthy and inflamed colonic mucosa of the DNBS-induced rat model. TfR expression was elevated in the colonic mucosa of IBD patients in both the basolateral and apical membranes of the enterocytes. Increased TfR expression was also observed in colonocytes of the induced colitis rats. To explore the underlying mechanism CaCo-2 cells were treated with various proinflammatory cytokines, which increased both TfR expression and transferrin cellular uptake in a mechanism that did not involve hyper proliferation. These findings were then exploited for the design of targetable carrier towards inflamed regions of the colon. Anti-TfR antibodies were conjugated to nano-liposomes. As expected, iron-starved Caco-2 cells internalized anti-TfR immunoliposomes better than controls. Ex vivo binding studies to inflamed mucosa showed that the anti-TfR immunoliposomes accumulated significantly better in the mucosa of DNBS-induced rats than the accumulation of non-specific immunoliposomes. It is concluded that targeting mucosal inflammation can be accomplished by nano-liposomes decorated with anti-TfR due to inflammation-dependent, apical, elevated expression of the receptor.

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t(1/2) ~1 h), or a slow, zero-order release rate (t(1/2) ~ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

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.

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.

The present study shows the advantages of liposome-based nano-drugs as a novel strategy of delivering active pharmaceutical ingredients for treatment of neurodegenerative diseases that involve neuroinflammation. We used the most common animal model for multiple sclerosis (MS), mice experimental autoimmune encephalomyelitis (EAE). The main challenges to overcome are the drugs' unfavorable pharmacokinetics and biodistribution, which result in inadequate therapeutic efficacy and in drug toxicity (due to high and repeated dosage). We designed two different liposomal nano-drugs, i.e., nano sterically stabilized liposomes (NSSL), remote loaded with: (a) a "water-soluble" amphipathic weak acid glucocorticosteroid prodrug, methylprednisolone hemisuccinate (MPS) or (b) the amphipathic weak base nitroxide, Tempamine (TMN). For the NSSL-MPS we also compared the effect of passive targeting alone and of active targeting based on short peptide fragments of ApoE or of β-amyloid. Our results clearly show that for NSSL-MPS, active targeting is not superior to passive targeting. For the NSSL-MPS and the NSSL-TMN it was demonstrated that these nano-drugs ameliorate the clinical signs and the pathology of EAE. We have further investigated the MPS nano-drug's therapeutic efficacy and its mechanism of action in both the acute and the adoptive transfer EAE models, as well as optimizing the perfomance of the TMN nano-drug. The highly efficacious anti-inflammatory therapeutic feature of these two nano-drugs meets the criteria of disease-modifying drugs and supports further development and evaluation of these nano-drugs as potential therapeutic agents for diseases with an inflammatory component.

PEGylated liposomal doxorubicin (Doxil) has been used in cancer chemotherapy for 16 years. Clinical experience shows that it can cause mild-to-severe hypersensitivity (infusion) reactions, which are manifestations of complement (C) activation-related pseudoallergy (CARPA). Although in most cases CARPA is inconsequential, a main symptom, cardiopulmonary distress, may be life threatening in hypersensitive individuals. To date, the prevention of Doxil-induced CARPA is based on premedication and a slow infusion protocol. The present study suggests desensitization by Doxil-like empty liposomes, called placebo Doxil (Doxebo), as an alternative strategy, which is based on the tachyphylactic nature of Doxil reactions. Doxebo-induced tolerance to Doxil was shown to develop within minutes and to be specific to Doxil-like PEGylated liposomes. The procedure of desensitization involves slow, low-dose pre-infusion of Doxebo before Doxil treatment which minimizes the ensuing physiological changes or keeps them subclinical. Although the mechanism of tolerance induction is not yet clear, the effector arm of C response is unlikely to be affected, as the vascular reactivity of desensitized pigs to zymosan remains intact. Desensitization with empty vesicles represents a novel approach for reducing the risk of anaphylactic reactions to drug carrier liposomes. The underlying immediate, most likely passive silencing of an innate immune response may represent a novel mechanism of tolerance induction which may work for other reactogenic nanosystems as well.

Cerebral malaria (CM) is a severe complication of and a leading cause of death due to Plasmodium falciparum infection. CM is likely the result of interrelated events, including mechanical obstruction due to parasite sequestration in the microvasculature, and upregulation of Th1 immune responses. In parallel, blood-brain-barrier (BBB) breakdown and damage or death of microglia, astrocytes, and neurons occurs. We found that a novel formulation of a liposome-encapsulated glucocorticosteroid, β-methasone hemisuccinate (nSSL-BMS), prevents experimental cerebral malaria (ECM) in a murine model and creates a survival time-window, enabling administration of an antiplasmodial drug before severe anemia develops. nSSL-BMS treatment leads to lower levels of cerebral inflammation, expressed by altered levels of corresponding cytokines and chemokines. The results indicate the role of integrated immune responses in ECM induction and show that the new steroidal nanodrug nSSL-BMS reverses the balance between the Th1 and Th2 responses in malaria-infected mice so that the proinflammatory processes leading to ECM are prevented. Overall, because of the immunopathological nature of CM, combined immunomodulator/antiplasmodial treatment should be considered for prevention/treatment of human CM and long-term cognitive damage.

Antitumor therapy in the elderly is particularly challenging due to multiple, often chronic diseases, poly-therapy, and age-related physiological changes that affect drug efficacy and safety. Furthermore, tumors may become more aggressive and drug-resistant with advanced age, leading to poor patient prognosis. In this study, we evaluated in mice bearing medulloblastoma xenografts the effect of age on tumor progression and tumor therapy. We focused on therapeutic efficacy of two treatment modalities alone radiofrequency ablation therapy (RFA), PEGylated liposomal doxorubicin (PLD) equivalent to Doxil, and their combination. We demonstrated that tumor growth rate was higher and survival was lower in old versus young mice (p<0.05). Likewise, tumors in old mice were less susceptible to either PLD or RFA monotherapy. However, combined therapy of PLD and RFA succeeded to eliminate the age-related differences in anti-cancer treatment efficacy (p>0.05) by the two monotherapies. The results on PLD therapy are supported by preferable PEGylated nano-liposomes accumulation in tumors of young mice compared to old mice, as determined by near-infrared imaging with indocyanine green (ICG)-labeled PEGylated nano-liposomes. Taken together, our findings suggest that age effects on tumor progression and tumor monotherapy outcome may potentially be related to changes in tumor microenvironment, and that these changes can be overcome by RFA as this technique abolishes these differences and significantly improves success of PLD treatment.

Staphylococcus aureus is a major cause of severe invasive infections. The increasing incidence of infections caused by antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA), calls for exploration of new approaches to treat these infections. Mupirocin is an antibiotic with a unique mode of action that is active against MRSA, but its clinical use is restricted to topical administration because of its limited plasma stability and rapid degradation to inactive metabolites. Mupirocin was identified by a machine learning approach to be suitable for nano-liposome encapsulation. The computational predictions were verified experimentally and PEGylated nano-liposomal formulation of mupirocin (Nano-mupirocin) was developed. The aim of this study was to investigate the efficacy of this formulation when administered parenterally for the treatment of S. aureus invasive infections. Nano-mupirocin exhibited prolonged half-life of active antibiotic and displayed superior antimicrobial activity against S. aureus than free mupirocin in the presence of plasma. Parenteral application of Nano-mupirocin in a murine model of S. aureus bloodstream infection resulted in improved antibiotic distribution to infected organs and in a superior therapeutic efficacy than the free drug. Parenterally administered Nano-mupirocin was also more active against MRSA than free mupirocin in a neutropenic murine lung infection model. In addition, Nano-mupirocin was very efficiently taken up by S. aureus-infected macrophages via phagocytosis leading to enhanced delivery of mupirocin in the intracellular niche and to a more efficient elimination of intracellular staphylococci. The outcome of this study highlights the potential of Nano-mupirocin for the treatment of invasive MRSA infections and support the further clinical development of this effective therapeutic approach.

Liposomes are employed as drug delivery vehicles offering a beneficial pharmacokinetic/distribution mechanism for in vivo therapeutics. Therapeutic liposomes can be designed to target specific cell types through the display of epitope-specific targeting peptides on their surface. The majority of peptides are currently attached by chemical modification of lipid constituents. Here we investigate an alternative and novel method of decorating liposomes with targeting ligand, using remotely and spontaneously inserting chimeric tail-anchored membrane (TA) proteins to drug loaded liposomes.

The uniqueness of Doxil can be attributed, to a large extent, to its intraliposomal doxorubicin-sulfate nanorod crystal. We re-examine these nanocrystal features and their mechanism of the formation by studying pegylated liposomal doxorubicins (PLDs) of the same lipid composition, size distribution, and extraliposome medium that were prepared at different ammonium sulfate (AS) concentrations. This study includes a comparison of the thermotropic behavior, morphology, and in vitro ammonia-induced doxorubicin release (relevant to Doxil's in vivo performance) of these PLDs. In this study, we confirm that a transmembrane ammonium gradient is critical for doxorubicin remote loading, and we demonstrate that the intraliposomal concentration of sulfate counteranions and ammonium ions determine to a large extent the physical state and stability of the PLDs' remote loaded doxorubicin. "Fully-developed" intraliposome doxorubicin-sulfate nanorod crystals (as defined by cryogenic transmission electron microscopy imaging) develop only when the ammonium sulfate (AS) concentration used for PLD preparation is ≥150 mM. Less than 10% of PLDs prepared with 100 mM AS show fully developed nanorod crystals. Intraliposomal AS concentration ≥200 mM is required to support the stable nanocrystallization in PLDs. The presence of nanocrystals and their melting enthalpy and phase transition co-operativity strongly affect the ammonia-induced doxorubicin release of PLDs. A quick, biphasic release occurs for PLDs that lack the nanorod crystals or have crystals of poor crystallinity, whereas PLDs prepared with ≥200 mM AS show a monophasic, zero-order slow release. This study also demonstrates that after remote loading, residual intraliposomal ammonium concentration and the transmembrane pH gradient related to it also play an important role in doxorubicin-sulfate intraliposomal crystallization and ammonia-induced doxorubicin release.

Artemisinins are the newest class of drug approved for malaria treatment. Due to their unique mechanism of action, rapid effect on Plasmodium, and high efficacy in vivo, artemisinins have become essential components of malaria treatment. Administration of artemisinin derivatives in combination with other anti-plasmodials has become the first-line treatment for uncomplicated falciparum malaria. However, their efficiency in cases of cerebral malaria (CM) remains to be determined.

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.

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.