Nanoparticles in liposomes: a platform for increased antibiotic selectivity in multidrug resistant bacteria in respiratory tract infections.

Antibiotic resistance is a cause of serious illness and death, originating often from insufficient permeability into gram-negative bacteria. Nanoparticles (NP) can increase antibiotic delivery in bacterial cells, however, may as well increase internalization in mammalian cells and toxicity. In this work, NP in liposome (NP-Lip) formulations were used to enhance the selectivity of the antibiotics (3C and tobramycin) and quorum sensing inhibitor (HIPS-1635) towards Pseudomonas aeruginosa by fusing with bacterial outer membranes and reducing uptake in mammalian cells due to their larger size. Poly (lactic-co-glycolic) acid NPs were prepared using emulsion solvent evaporation and incorporated in larger liposomes. Cytotoxicity and uptake studies were conducted on two lung cell lines, Calu-3 and H460. NP-Lip showed lower toxicity and uptake in both cell lines. Then formulations were investigated for suitability for oral inhalation. The deposition of NP and NP-Lip in the lungs was assessed by next generation impactor and corresponded to 75% and 45% deposition in the terminal bronchi and the alveoli respectively. Colloidal stability and mucus-interaction studies were conducted. NP-Lip showed higher diffusion through mucus compared to NPs with the use of nanoparticle tracking analyzer. Moreover, the permeation of delivery systems across a liquid-liquid interface epithelial barrier model of Calu-3 cells indicated that NP-Lip could cause less systemic toxicity upon in-vivo like administration by aerosol deposition. Monoculture and Pseudomonas aeruginosa biofilm with Calu-3 cells co-culture experiments were conducted, NP-Lip achieved highest toxicity towards bacterial biofilms and least toxicity % of the Calu-3 cells. Therefore, the NP- liposomal platform offers a promising approach for enhancing antibiotic selectivity and treating pulmonary infections.

A liposomal platform for the delivery of ion channel proteins for treatment of channelopathies - Application in therapy of cystic fibrosis.

Channelopathies arise from ion channel dysfunction. Successful treatment entails delivery of functional ion channels to replace dysfunctional ones. Glycine receptor (GlyR)-rich cell membrane fragments (CMF) were previously delivered to target cell membranes using fusogenic liposomes. Here, cystic fibrosis transmembrane conductance regulator (CFTR)-bearing CMF were similarly delivered to target cells. We studied the effect of lipid composition on liposomes' ability to incorporate CMF and fuse with target cell membranes to deliver functional CFTR. Four formulations were prepared using thin-film hydration out of different lecithin sources, egg and soy lecithin (EL and SL), in the presence and absence of cholesterol (CHOL): EL + CHOL, EL-CHOL, SL + CHOL, and SL-CHOL. EL liposomes incorporated more CMF than SL liposomes, with CHOL only increasing CMF incorporation in SL liposomes. SL + CHOL fused better with target cell membranes than EL + CHOL. SL + CHOL and EL + CHOL equally delivered CFTR to target cell membranes, owing to the former's superior fusogenic capacity and the latter's superior CMF-incorporation capacity. SL-CHOL and EL-CHOL delivered CFTR to a lesser extent, indicating the importance of CHOL for fusion. Patch-clamp electrophysiology and confocal laser scanning microscopy (CLSM) confirmed CFTR delivery to target cell membranes by SL + CHOL. Therefore, CMF-bearing fusogenic liposomes offer a promising universal platform for the treatment of channelopathies.

A Review of Current and Prospective Treatments for Channelopathies, with a Focus on Gene and Protein Therapy.

Reduced cell surface expression or the malfunctioning of ion channels gives rise to a group of disorders known as channelopathies. To treat the underlying cause, the delivery and/or expression of a functional ion channel into the cell membrane of the cell of interest is required. Unfortunately, for most channelopathies, current treatment options are only symptomatic and treatments that rectify the underlying damage are still lacking. Within this context, approaches that rely on gene and protein therapy are required. Gene therapy would allow the expression of a functional protein, provided that the cellular machinery in the diseased cell could correctly fold and traffic the protein to the cell membrane. Whereas protein therapy would allow the direct delivery of a functional protein, provided that the purification process does not affect protein function and a suitable delivery vehicle for targeted delivery is used. In this review, we provide an overview of channelopathies and available symptomatic treatments. The current state of gene therapy approaches mainly using viral vectors is discussed, which is followed by the role of nanomedicine in protein therapy and how nanomedicine could be exploited for the delivery of functional ion channels to diseased cells.

Enhancement of mitochondrial function using NO releasing nanoparticles; a potential approach for therapy of Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia. Increasing evidence is showing the important role of mitochondrial dysfunction in AD. Mitochondria based oxidative stress, decrease in respiratory chain activity and ATP production are all associated with AD, hence indicating that the enhancement of mitochondrial function and biogenesis present a promising therapeutic approach for AD. Nitric oxide (NO) is an initiator of mitochondrial biogenesis. However, its gaseous nature and very short half-life limit the realization of its therapeutic potential. Additionally, its uncontrolled in-vivo distribution results in generalized vasodilation, hypotension among other off-target effects. Diazeniumdiolates (NONOates) are NO donors that release NO in physiological temperature and pH. Their encapsulation within a hydrophobic matrix carrier system could control the release of NO, and at the same time enable its delivery to the brain. In this work, PAPANONOate (PN) a NO donor was encapsulated in small (92 ± 7 nm) poly (lactic-co-glycolic acid) (PLGA) NPs. These NPs did not induce hemolysis upon intravenous administration and were able to accumulate in the brains of lipopolysaccharides (LPS) induced neurodegeneration mouse models. The encapsulation of PN within a hydrophobic PLGA matrix enabled the sustained release of NO from NPs (≈ 3 folds slower relative to free PN) and successfully delivered PN to brain. As a result, PN-NPs but not free PN resulted in an enhancement in memory and cognition in animals with neurodegeneration as determined by the Y-maze test. The enhancement in cognition was a result of increased mitochondria function as indicated by the increased production of ATP and Cytochrome C oxidase enzyme activity.

Drugless nanoparticles tune-up an array of intertwined pathways contributing to immune checkpoint signaling and metabolic reprogramming in triple-negative breast cancer.

Metabolic reprogramming 'Warburg effect' and immune checkpoint signaling are immunosuppressive hallmarks of triple-negative breast cancer (TNBC) contributing to the limited clinical applicability of immunotherapy. Biomaterials arise as novel tools for immunomodulation of the tumor microenvironment that can be used alongside conventional immunotherapeutics. Chitosan and lecithin are examples of versatile biomaterials with interesting immunomodulatory properties. In this study, we aimed at investigation of the role of carefully designed hybrid nanoparticles (NPs) on common mediators of both programmed death ligand 1 (PD-L1) expression and glycolytic metabolism. Hybrid lecithin-chitosan NPs were prepared and characterized. Their intracellular concentration, localization and effect on the viability of MDA-MB-231 cells were assessed. Glycolytic metabolism was quantified by measuring glucose consumption, adenosine triphosphate (ATP) generation, lactate production and extracellular acidification. Nitric oxide production was quantified using Greiss reagent. Gene expression of inducible nitric oxide synthase (iNOS), phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB or Akt), mammalian target of rapamycin (mTOR), hypoxia-inducible factor 1α(HIF-1α) and PD-L1 was quantified by quantitative reverse transcription polymerase chain reaction (q-RT-PCR). Chitosan, lecithin and the NPs-formulated forms have been shown to influence the 'Warburg effect' and immune checkpoint signaling of TNBC cells differently. The composition of the hybrid systems dictated their subcellular localization and hence the positive or negative impact on the immunosuppressive characteristics of TNBC cells. Carefully engineered hybrid lecithin-chitosan NPs could convert the immune-suppressive microenvironment of TNBC to an immune-active microenvironment via reduction of PD-L1 expression and reversal of the Warburg effect.

Immunosuppressive Effect of Intrathecal Morphine, Dexmedetomidine, or Both in Combination with Bupivacaine on Patients Undergoing Major Abdominal Cancer Surgery.

BACKGROUND: An impaired immune system in the perioperative period has important clinical implications in patients with cancer. Despite the immunosuppressive properties of opioid therapy, it is still commonly utilized in the intrathecal or epidural space for the treatment of postoperative pain. Also, intrathecal dexmedetomidine has extended analgesic efficacy in postoperative pain; it can significantly affect immune function in perioperative patients. OBJECTIVE: To investigate the effect of intrathecal morphine, dexmedetomidine, or both in combination with bupivacaine on cellular immunity and cytokine production in cancer surgical patients. STUDY DESIGN: A prospective randomized clinical study. SETTING: South Egypt Cancer Institute, Assiut University. METHODS: Ninety patients were randomly assigned to receive intrathecal morphine 0.5 mg (Group M, n = 30), dexmedetomidine 0.5 µg/kg (Group D, n = 30) or morphine 0.5 mg with dexmedetomidine 0.5 µg/kg (Group MD n = 30); 2 mL bupivacaine 0.5% was added to injected drugs in all groups.  Blood samples were collected preoperative (T0), immediate postoperative (T1), 4 hours postoperative (T2), and 24 hours postoperative (T3) for measurement of CD3, CD4, CD4/CD8 and CD16+56(NK), interleukin(IL)-1beta (IL-1beta), IL-6, IL-10 and tumor necrosis factor alpha (TNF-alpha). RESULTS: A significant reduction in cellular immunity (CD3, CD4, CD8, CD4/CD8, CD 16+56) was noticed in the 24-hour postoperative period in all 3 studied groups, with a marked reduction in Group M in comparison to Group MD and Group D. Regarding inflammatory mediators, IL-10 and IL-1beta  showed significant reduction in Group M in the first 24-hour postoperative period in comparison to Group MD and Group D, while IL-6 was significantly reduced in Group MD and Group D in comparison to Group M in the same period. TNF-alpha was significantly increased postoperative at T1 and T2 in the 3 studied groups, then at T3 it decreased without a statistically significant difference with the preoperative level. LIMITATIONS: Our study has some limitations, such as the short period of follow-up and lack of postoperative clinical follow-up of patients to discover the association between immunity and patient outcomes. CONCLUSION: Intrathecal dexmedetomidine has the least immunosuppressive effect than morphine and morphine-dexmedetomidine, in combination with bupivacaine.

The skin delivery of tofacitinib citrate using transethosomes and hybridized ethosomes/nanostructured lipid carriers for vitiligo therapy: Dermatopharmacokinetics and in vivo assays.

Vitiligo is an autoimmune disease where its current treatment strategies are lengthy in course and do not guarantee complete cure. Tofacitinib citrate (JAK inhibitor) is a potential cure of vitiligo through halting JAK-STAT pathway preventing the destruction of melanocytes. The dermato-pharmacokinetics of the prepared transethosomes (Et) and the hybridized ethosomes/nanostructured lipid carriers (Eth/NLC), namely formulations; M.E-Cr and M.E-S.M, were evaluated. In addition, in vivo studies on C57/BL6 vitiligo mouse model were conducted to confirm effectiveness of Tofacitinib citrate delivery. The results unveiled that the transethosomes (359.46 ± 11.82 nm) were suitable for dermal delivery while M.E-Cr (179.64 ± 11.16 nm), a hybrid Eth/NLC formulation, was mostly suitable for transdermal delivery. Nevertheless, another hybrid formulation, M.E-S.M (253.60 ± 14.64 nm), was apt for both dermal and transdermal delivery. The histopathology confirmed re-pigmentation of mice skin where formulations Et and M.E-S.M showed severe pigmentation compared to the control healthy and induced mice. On the other hand, M.E-Cr showed mild pigmentation. Immunohistochemical assay was performed to evaluate infiltration of CD 8+T-lymphocytes where mild infiltration was observed. However, the systemic IFN-γ was significantly reduced in case of M.E-Cr and M.E-S.M. The present work proposed potential effective formulations to improve the treatment of vitiligo with potential reduction in the total therapeutic dose, drug's side effects, and treatment costs.

Nanoparticle Fraught Liposomes: A Platform for Increased Antibiotic Selectivity in Multidrug Resistant Bacteria.

Increasing antibiotic concentrations within bacterial cells while reducing them in mammalian ones would ultimately result in an enhancement of antibacterial actions, overcoming multidrug resistance, all while minimizing toxicity. Nanoparticles (NPs) have been used in numerous occasions to overcome antibiotic resistance, poor drug solubility, and stability. However, the concomitant increase in antibiotic concentration in mammalian cells and the resultant toxicity are usually overlooked. Without compromising bacterial cell fusion, large liposomes (Lip) have been reported to show reduced uptake in mammalian cells. Therefore, in this work, small NP fraught liposomes (NP-Lip) were formulated with the aim of increasing NP uptake and antibiotic delivery in bacterial cells but not in mammalian ones. Small polylactic-co-glycolic acid NPs were therefore loaded with erythromycin (Er), an antibiotic with low membrane permeability that is susceptible to drug efflux, and 3c, a 5-cyanothiazolyl urea derivative with low solubility and stability. In vitro experiments demonstrated that the incorporation of small NPs into large Lip resulted in a reduction in NP uptake by HEK293 cells while increasing it in Gram-negative bacteria (Escherichia coli DH5α, E. coli K12, and Pseudomonas aeruginosa), consequently resulting in an enhancement of antibiotic selectivity by fourfold toward E. coli (both strains) and eightfold toward P. aeruginosa. Ocular administration of NP-Lip in a P. aeruginosa keratitis mouse model demonstrated the ability of Er/3c-loaded NP-Lip to result in a complete recovery. More importantly, in comparison to NPs, the ocular administration of NP-Lip showed a reduction in TNF-alpha and IL-6 levels, implying reduced interaction with mammalian cells in vivo. This work therefore clearly demonstrated how tailoring the nano-bio interaction could result in selective drug delivery and a reduction in toxicity.

T-lymphocytes Expression of Toll-like Receptors 2 and 4 in Acute Myeloid Leukemia Patients with Invasive Fungal Infections.

Background: Invasive fungal infections (IFIs) are important cause of mortality in acute myeloid leukemia (AML) patients on treatment with intensive induction chemotherapy. Toll-like receptors, mainly Toll-like receptors 2 and 4 (TLR2 and TLR4), play a considerable role in the host defense against microorganisms. The involvement of TLR signaling in modulation of innate immune response is extensively discussed, but the TLR expressions profiling on adaptive immune cells are not specified. Also, the expressions of TLRs and their association with the occurrence of IFIs in patients with AML are not studied. So, the novel aim of this study was to investigate the associations between the T-lymphocyte expression of TLR2 and TLR4 and the occurrence of IFIs in AML patients treated with intensive induction chemotherapy. Materials and Methods: One hundred twenty two newly diagnosed AML patients were evaluated. The laboratory diagnostic techniques for IFIs include culture, microscopic examination, histopathology, galactomannan assay and PCR. The expressions of TLR2 and TLR4 were analyzed by flow cytometry. The Control group included 20 age and sex-matched individuals. Results: There was a significant increase in the expression of TLR4 in AML patients with IFI compared to healthy controls (p = 0.001). TLR2 and TLR4 expressions increased significantly in AML patients with mixed fungal and bacterial infection compared to healthy controls (p= 0.002 and p=0.001, respectively). Conclusion: TLRs expressions could be important biological markers for the occurrence of IFI in non-M3 AML patients after intensive induction chemotherapy.

Different Serum, Different Protein Corona! The Impact of the Serum Source on Cellular Targeting of Folic Acid-Modified Chitosan-Based Nanoparticles.

The nanoparticle (NP) protein corona represents an interface between biological components and NPs, dictating their cellular interaction and biological fate. To assess the success of cellular targeting, NPs modified with targeting ligands are incubated with target cells in serum-free culture medium or in the presence of fetal bovine serum (FBS). In the former, the role of the corona is overlooked, and in the latter, the effects of a corona that does not represent the one forming in humans nor the respective disease state are considered. Via proteomic analysis, we demonstrate how the difference in the composition of FBS, sera from healthy human volunteers, and breast cancer patients (BrCr Pt) results in the formation of completely different protein coronas around the same NP. Successful in vitro targeting of breast cancer cells was only observed when NPs were incubated with target cells in the presence of BrCr Pt sera only. In such cases, the success of targeting was not attributed to the targeting ligand itself, but to the adsorption of specific serum proteins that facilitate NP uptake by cancer cells in the presence of BrCr Pt sera. This work therefore demonstrates how the serum source affects the reliability of in vitro experiments assessing NP-cell interactions and the consequent success or failure of active targeting and may in fact indicate an additional reason for the limited clinical success of drug targeting by NPs in cancer.

Experiences of first-line nurse managers during COVID-19: A Jordanian qualitative study.

AIM: The aim of this study is to explore the experiences of Jordanian first-line nurse managers during COVID-19. BACKGROUND: Nurses are exposed to life-threatening occupational risks during COVID-19. Exploring the first-line nurse managers' experiences will help in designing health policies to better deal with such emerging crises. METHODS: A descriptive phenomenological study was conducted. A purposive sample was used to recruit 16 first-line nurse managers from Jordanian hospitals. Semistructured interviews were conducted. Phenomenological data analysis method was used to analyse the data. RESULTS: Four major themes emerged: (a) unprecedented pressure (first-line nurse managers revealed their suffering with the unprecedented demanding situations during COVID-19 pandemic); (b) strengthening system and resilience (nurse managers employed several strategies to strengthen the health system and enhance resilience); (c) building a supportive team (the presence of a robust supportive system is vital to deal with the pandemic); and (d) maturity during the crisis (exposure to a new experience developed nurse managers management skills and self-awareness). CONCLUSIONS: The unprecedented pressure associated with COVID-19 drained first-line nurse managers physically and psychosocially. Providing adequately trained staff and medical equipment is important to better deal with crises. IMPLICATIONS FOR NURSING MANAGEMENT: Strengthening emergency training and improving emergency response plans of hospitals are essential.

The other side to the use of active targeting ligands; the case of folic acid in the targeting of breast cancer.

Due to its overexpression in cancer cells, the folate receptor (FR) is heavily exploited in the active targeting of nanoparticles (NPs). Its ligand, folic acid (FA) is as a consequence widely used as a NP targeting ligand. Although rather popular and successful in principle, recent data has shown that FA may result in breast cancer initiation and progression, which questions the suitability of FA as NP cancer targeting ligand. In this work, intravenous administration of free FA to healthy female mice resulted in breast tissue dysplasia, hyperplasia and in the increased expression of human epidermal growth factor receptor-2 (HER2), folate receptor (FR), cancer antigen 15-3 (CA15.3), vascular endothelial growth factor (VEGF), signal transducer and activator of transcription 3 (STAT3) and the pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6) and interleukin-1ß. In addition to the reduction in IL2. To evaluate the suitability and safety of FA as NP targeting ligand in breast cancer, small (≈ 150 nm) and large (≈ 500 nm) chitosan NPs were formulated and decorated with two densities of FA. The success of active targeting by FA was confirmed in two breast cancer cell lines (MCF-7 and MDA-MB-231 cells) in comparison to HEK293 cells. FA modified NPs that demonstrated successful active targeting in-vitro were assessed in-vivo. Upon intravenous administration, large NPs modified with a high density of FA accumulated in the breast tissue and resulted in similar effects as those observed with free FA. These results therefore question the suitability of FA as a targeting ligand in breast cancer and shed light on the importance of considering the activity (other than targeting) of the ligands used in NP active targeting.

Delivery of trans-membrane proteins by liposomes; the effect of liposome size and formulation technique on the efficiency of protein delivery.

Channelopathies are disorders caused by reduced expression or impaired function of ion channels. Most current therapies rely on symptomatic treatment without addressing the underlying cause. We have recently established proof of principle for delivery of functional ion channel protein into the membrane of target cells using fusogenic liposomes incorporating glycine receptor (GlyR)-containing cell membrane fragments (CMF) that were formulated by thin film hydration. Here, the effect of liposome size and the formulation technique on the performance of the delivery vehicle was assessed. Three types of liposomes were prepared using lecithin and cholesterol, (i) small (SL), and (ii) large (LL) liposomes made by thin film hydration, and (iii) small liposomes prepared by vortex agitation (V-SL). All liposomes were evaluated for their ability to (i) incorporate GlyR-rich CMF, (ii) fuse with the cell membrane of target cells and (iii) deliver functional GlyR, as assessed by patch-clamp electrophysiology. SL prepared by thin film hydration offered the most effective delivery of glycine receptors that gave clear glycine-mediated currents in target cells. LL showed higher incorporation of CMF, but did not effectively fuse with the target cell membrane, while V-SL did not incorporate sufficient amounts of CMF. Additionally, SL showed minimalin vivotoxicity upon intrathecal injection in mice. Thus, liposome-mediated delivery of membrane proteins may be a promising therapeutic approach for the treatment of channelopathies.

Gamma sterilization and in vivo evaluation of cationic nanostructured lipid carriers as potential ocular delivery systems for antiglaucoma drugs.

Solid lipid nanoparticles and nanostructured lipid carriers showed promising results for enhancement of ocular bioavailability of drugs with poor corneal permeability. One of these drugs is methazolamide, which is an orally administered carbonic anhydrase inhibitor for glaucoma treatment. However, sterilization by autoclaving may result in loss of the physical properties of lipid nanoparticles such as particle size and surface charge. Here, we evaluated gamma radiation as an alternative sterilization method. Methazolamide loaded nanostructured lipid carriers were optimized using 23 factorial design. Optimized formulations contained 6% lipid (85% solid lipid (Cetostearyl alcohol and glyceryl behenate) and 15% oil either medium chain triglycerides or isopropyl myristate) stabilized by 2% polysorbate 80 and 0.15% stearylamine. Nanoparticles were cationic, smaller than 500 nm, and had an entrapment efficiency of about 30%. They released methazolamide within 8 hours and showed a 5-fold enhanced reduction in intraocular pressure compared to methazolamide solution. Gamma sterilization was superior to autoclaving in preserving entrapped methazolamide, size, and surface charge of lipid nanoparticles. These findings demonstrate that gamma radiation is a viable alternative to autoclaving for sterilizing lipid nanoparticles. Moreover, this proves that nanostructured lipid carriers enhance pharmacological response of topically administered methazolamide for treating glaucoma.

Targeting Activated Hepatic Stellate Cells Using Collagen-Binding Chitosan Nanoparticles for siRNA Delivery to Fibrotic Livers.

Activated hepatic stellate cells (aHSCs) are the main orchestrators of the fibrotic cascade in inflamed livers, with transforming growth factor-beta (TGF-ß) being the most potent pro-fibrotic cytokine. Hence, aHSCs serve as interesting therapeutic targets. However, drug delivery to aHSCs is hindered by excessive collagen deposition in the extracellular matrix (ECM) and capillarization of liver sinusoids. Chitosan-nanoparticles (CS-NPs) show intrinsic affinity for collagen, holding potential for drug delivery to fibrotic livers. Here, we employed CS-NPs for anti-TGF-ß siRNA delivery, promoting delivery into aHSCs via modification with platelet-derived growth factor receptor-beta binding peptides. In-vitro experiments using aHSCs demonstrated the association of unmodified CS-NPs to the collagen-rich ECM, with reduced intracellular accumulation. Peptide-modified CS-NPs showed a higher propensity to localize intracellularly; however, this was only the case upon ECM-collagen reduction via collagenase treatment. Peptide-modified CS-NPs were more potent than unmodified CS-NPs in reducing TGF-ß expression, implying that while collagen binding promotes liver accumulation, it hinders cell-specific siRNA delivery. In-vivo, CS-NPs successfully accumulated in fibrotic livers via collagen binding. Similar to in-vitro findings, when mice were pretreated with collagenase-loaded CS-NPs, the accumulation of peptide-modified NPs increased. Our findings demonstrate the usefulness of NPs modification with targeting ligands and collagenase treatment for aHSCs targeting and highlight the importance of chitosan-collagen binding in drug delivery to fibrotic diseases.

Collagenase loaded chitosan nanoparticles for digestion of the collagenous scar in liver fibrosis: The effect of chitosan intrinsic collagen binding on the success of targeting.

A variety of hepatic insults result in the accumulation of collagen-rich new extracellular matrix in the liver, ultimately culminating in liver fibrosis and cirrhosis. For such reasons, approaches looking into digestion of the collagen-rich extracellular matrix present an interesting therapeutic approach for cases of chronic liver disease, where the fibrotic scar is well established. Portal collagenase administration has recently led to the successful reversion of cirrhosis in an experimental rabbit model. Notwithstanding, the question of how such a sensitive therapeutic macromolecule could be administered in a less invasive manner, and in a way that preserves its functionality and avoids digestion of other non-hepatic vital collagen presents itself. Chitosan is a biodegradable polymer that has been reported to interact and bind to collagen. Chitosan nanoparticles (CS NPs) have also been reported to encapsulate therapeutic proteins, maintaining their functional form and protecting them from in-vivo degradation. For such reasons, CS NPs were loaded with collagenase and evaluated in-vitro and in-vivo for their ability to target and digest collagen. CS NPs were able to encapsulate collagenase (≈ 60% encapsulation efficiency) and release its content in active form. To determine whether chitosan's collagen interaction would enable NP collagen binding or whether the modification with collagen binding peptides (CBPs) is necessary, CS NPs were modified with the CBP; CCQDSETRTFY. Since the density of targeting ligand and the length of tether play a significant role in the success of active targeting, the surface of NPs was modified with different densities of the CBP either directly or using a polyethylene glycol (PEG) spacer. PEGylated NPs showed higher levels of CBP tagging; high, intermediate and low density of CBPs corresponded to 585.8 ± 33, 252.9 ± 25.3 and 56.5 ± 8.8 µg/mL for PEGylated NPs and 425.56 ± 12.67, 107.91 ± 10.3 and 49.86 ± 3.2 µg/mL for unPEGylated NPs, respectively. In-vitro collagen binding experiments showed that unmodified CS NPs were able to bind collagen and that modification with CBPs either directly or via PEG did not enhance collagen binding. In-vivo experiments demonstrated that unmodified CS NPs were able to reverse fibrosis with a survival rate of 100% at the end of the study, indicating the ability of CS NPs to deliver functional collagenase to the fibrotic liver and making the use of CBPs unnecessary.

Liposomal delivery of functional transmembrane ion channels into the cell membranes of target cells; a potential approach for the treatment of channelopathies.

Defects in transmembrane ion channels underlie many disorders, commonly known as channelopathies. Current therapies are mostly symptomatic and do not treat the underlying cause. Here, we demonstrate the delivery of functional ion channels in protein form into the membrane of target cells using fusogenic proteoliposomes. The glycine receptor (GlyR) was adopted as a model channel. HEK293 cells were transfected with GlyR and GlyR-rich cell membrane fragments (CMF) were incorporated into fusogenic liposomes. Proteoliposomes were generated using 1,2-dioleoylphosphoethanolamine (DOPE) as the fusogenic lipid, lecithin, 1,2-distearoylphosphoethanolamine (DSPE), and cholesterol (Chol). Three formulations were prepared Non-fuse (2.5:0.5 Lecithin: Chol), Fuse1 (1.25:0.25:0.25:0.25) and Fuse2 (1.25:0.5:0.5:0.25 Lecithin: DOPE: DSPE: Chol). Proteoliposomes were assessed for their ability to (1) incorporate GlyR rich CMF (2) fuse with L929 fibroblast cell membrane and (3) deliver functional GlyR to these cells. All formulations were capable of integrating CMF, with Fuse2 showing highest CMF incorporation (1.2 and 1.4 folds relative to Non-fuse and Fuse1 respectively). All liposomes showed ability to fuse with the fibroblast cell membrane, with Fuse2 showing highest fusion. Patch-clamp analysis demonstrated successful delivery of functional GlyR into the fibroblast cell membrane. Thus, proof of principle was established for the use of liposomes to deliver functional ion channels to living cells.

Expression analysis of two SLAM family receptors, SLAMF2 and SLAMF7, in patients with multiple myeloma.

Monoclonal antibodies against surface antigens on MM cells, such as anti-SLAMF7 and anti-CD38 antibodies, represent an attractive therapeutic modality for the eradication of multiple myeloma (MM) cells. However, further exploration of target molecules is urgently needed for the development of more effective therapies. In the present study, we studied the expression of CD48 in a total of 74 primary MM samples derived from patients to evaluate SLAMF2 (CD48) as a candidate in mAb therapy for MM. Of 74 samples, 39 were subjected to SLAMF7 analysis. Most of the MM cells, defined as CD38 and CD138 double-positive cells, showed strong expression of CD48 or SLAMF7 independent of disease stage or treatment history. In these 39 samples, most MM cells showed expression of both SLAMF7 and CD48; however, several samples showed expression of either only CD48 or only SLAMF7, including seven cases that were only highly positive for SLAMF7, and five that were only highly positive for CD48. Our study demonstrates that the immune receptor CD48 is overexpressed on MM cells together with SLAMF7, and that CD48 may be considered as an alternative target for treatment of MM in cases showing weak expression of SLAMF7.

Effect of Preperitoneal Versus Epidural Analgesia on Postoperative Inflammatory Response and Pain Following Radical Cystectomy: A Prospective, Randomized Trial.

OBJECTIVES: Continuous wound infiltration of local anesthetics has been proposed as an alternative to epidural analgesia during abdominal surgery. Cytokines have a major role in inflammatory changes caused by surgery. This study aimed to compare the effects of continuous preperitoneal versus epidural analgesia on inflammatory cytokines postoperatively. MATERIALS AND METHODS: Forty patients scheduled for radical cystectomy were included in this observer-blinded, randomized trial; patients were randomly assigned into 2 groups to receive; continuous preperitoneal wound infiltration (PPB) or epidural analgesia (EDB). Serum levels of interleukins (IL1ß, IL6, IL10, and tumor necrosis factor α) were measured at baseline (before induction of anesthesia), preinfusion (before the start of local anesthetic infusion), 6 and 24 hours postoperatively. Visual Analog Scale at rest/movement (VAS-R/M), time to the first request of analgesia, total morphine consumption, sedation score, hemodynamics, and side effects were observed 24 hours postoperatively. RESULTS: There was a significant reduction in IL6, IL1ß and increase in IL10 in PPB compared with EDB at 6 and 24 hours postoperatively and compared with preinfusion levels (P≤0.001). In EDB, a significant increase in IL1ß, IL10, and tumor necrosis factor α at 6 hours compared with preinfusion levels (P≤0.002). VAS-R/M was significantly decreased at 2, 4, 6, 8, and 12 hours in EDB compared with PPB (P≤0.014), with no significant difference in the mean time to the first request of analgesia and total morphine consumption between the 2 groups. CONCLUSION: Continuous preperitoneal analgesia better attenuated postoperative inflammatory response and provided a comparable overall analgesia to that with continuous epidural analgesia following radical cystectomy.

Tailoring novel soft nano-vesicles 'Flexosomes' for enhanced transdermal drug delivery: Optimization, characterization and comprehensive ex vivo - in vivo evaluation.

The transdermal route is a convenient non-invasive way for drug delivery, however, the hydrophobic compact nature of stratum corneum (SC) forms an obstacle hindering the diffusion of drugs particularly hydrophilic ones. Hence, the purpose of this study was to develop novel soft nano-vesicles, entitled Flexosomes, amalgamating two penetration enhancers, ethanol and one edge activator (EA) from various types and different hydrophilic-lipophilic balances. The tailored vesicles were loaded with tropisetron hydrochloride (TRO), a potent highly-soluble anti-emetic, and compared with ethosomes. Aiming to preclude the formation of rigid non-deformable mixed micelles, all critical parameters; EA type, phosphatidylcholine-to-EA molar ratio, and cholesterol concentration, were optimized proving their influences on vesicle-to-micelle transitions. The prepared formulations were characterized in terms of visual inspection, particle size, polydispersity, zeta potential, turbidity measurements, entrapment efficiency, and vesicle morphology. The permeation mechanisms were assessed by differential scanning calorimetry on isolated SC. The modified vesicles, based on ethanol and either vitamin E or PEGylated castor oil derivatives exhibited the highest transdermal fluxes confirmed by a deeply tracking to dermis using confocal laser microscopy. Both vesicles demonstrated higher bioavailability relative to ethosomes, topical and oral aqueous solutions. The findings endorsed the effectiveness of tailored nano-vesicles in boosting TRO skin transport suggesting their applicability with various drug entities for enhanced transdermal delivery.