Vibhudutta Awasthi

Surface engineering of liposomes for stealth behavior.

Liposomes are used as a delivery vehicle for drug molecules and imaging agents. The major impetus in their biomedical applications comes from the ability to prolong their circulation half-life after administration. Conventional liposomes are easily recognized by the mononuclear phagocyte system and are rapidly cleared from the blood stream. Modification of the liposomal surface with hydrophilic polymers delays the elimination process by endowing them with stealth properties. In recent times, the development of various materials for surface engineering of liposomes and other nanomaterials has made remarkable progress. Poly(ethylene glycol)-linked phospholipids (PEG-PLs) are the best representatives of such materials. Although PEG-PLs have served the formulation scientists amazingly well, closer scrutiny has uncovered a few shortcomings, especially pertaining to immunogenicity and pharmaceutical characteristics (drug loading, targeting, etc.) of PEG. On the other hand, researchers have also begun questioning the biological behavior of the phospholipid portion in PEG-PLs. Consequently, stealth lipopolymers consisting of non-phospholipids and PEG-alternatives are being developed. These novel lipopolymers offer the potential advantages of structural versatility, reduced complement activation, greater stability, flexible handling and storage procedures and low cost. In this article, we review the materials available as alternatives to PEG and PEG-lipopolymers for effective surface modification of liposomes.

CLEFMA—An anti-proliferative curcuminoid from structure–activity relationship studies on 3,5-bis(benzylidene)-4-piperidones

3,5-Bis(benzylidene)-4-piperidones are being advanced as synthetic analogs of curcumin for anti-cancer and anti-inflammatory properties. We performed structure-activity relationship studies, by testing several synthesized 3,5-bis(benzylidene)-4-piperidones for anti-proliferative activity in lung adenocarcinoma H441 cells. Compared to the lead compound 1, or 3,5-bis(2-fluorobenzylidene)-4-piperidone, five compounds were found to be more potent (IC(50) < 30 microM), and 16 compounds possessed reduced cell-killing efficacy (IC(50) > 50 microM). Based on the observations, we synthesized 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] (29 or CLEFMA) as a novel analog of 1. CLEFMA was evaluated for anti-proliferative activity in H441 cells, and was found to be several folds more potent than compound 1. We did not find apoptotic cell population in flow cytometry, and the absence of apoptosis was confirmed by the lack of caspase cleavage. The electron microscopy of H441cells indicated that CLEFMA and compound 1 induce autophagic cell death that was inhibited by specific autophagy inhibitor 3-methyladenine. The results suggest that the potent and novel curcuminoid, CLEFMA, offers an alternative mode of cell death in apoptosis-resistant cancers.

Improved formulation of liposome-encapsulated hemoglobin with an anionic non-phospholipid.

We are developing liposome-encapsulated hemoglobin (LEH) as an artificial oxygen carrier for resuscitation in indications, such as acute blood loss and surgery. Earlier attempts to formulate a viable LEH met with constraints of scale up and limited hemoglobin content. In this work, we report an LEH formulation containing novel anionic non-phospholipid (CHHDA) that enhances the encapsulation efficiency of hemoglobin inside the liposome bilayer. CHHDA was synthesized from inexpensive ingredients in high yields. The formulation was evaluated in vitro to investigate the cytotoxic effects on RAW 264.7 macrophages and HUVEC endothelial cells in culture by LDH, MTT and hexosaminidase assays. Under optimal conditions of manufacturing, the presence of 28 mol% of CHHDA enhanced the hemoglobin content to over 4 g/dl. The LEH containing CHHDA shows some cytotoxicity in HUVEC and RAW cells in vitro, especially by LDH assay. MTT assay was negative for cytotoxicity in both cells lines. By hexosaminidase assay, the proliferation of RAW cells, but not HUVEC cells, was inhibited. When CHHDA-LEH was incubated with isolated human platelets in vitro, no platelet activation was observed. The LEH formulation with novel anionic lipid and high hemoglobin content reported in this article is an improvement from the past preparations.

Post-modification of preformed liposomes with novel non-phospholipid poly(ethylene glycol)-conjugated hexadecylcarbamoylmethyl hexadecanoic acid for enhanced circulation persistence in vivo

We report synthesis and characterization of a novel PEG2000-conjugated hexadecylcarbamoylmethyl hexadecanoate (HDAS-PEG) as a PEG-phospholipid substitute for enhancing circulation persistence of liposomes. HDAS-PEG showed critical micelle concentration of 4.25 μM. We used post-insertion technique to introduce HDAS-PEG in outer lipid layer of the preformed liposomes. The presence of surface HDAS-PEG was confirmed by altered electrophoretic mobility, confocal microscopy and PEG estimation by ELISA. The post-inserted HDAS-PEG desorbed at approximately half the rate at which post-inserted DSPE-PEG desorbed from the liposome surface. HDAS-PEG significantly reduced liposome-induced complement activation (C4d, Bb and SC5b); HDAS-PEG was more effective than more commonly used DSPE-PEG in this capacity. For studying circulation persistence, the liposomes were labeled with (99m)Tc radionuclide and administered in rats. (99m)Tc-HDAS-PEG-liposomes showed prolonged persistence in blood as compared to that shown by (99m)Tc-plain liposomes. After 24 h of administration, <1% of (99m)Tc-plain liposomes remained in blood, whereas approximately 28% of injected (99m)Tc-HDAS-PEG-liposomes were present in blood. In comparison, only 4.8% of (99m)Tc-DSPE-PEG-liposomes were measured in blood after 24 h. As expected, the clearance route of the liposomes was through liver and spleen. These results demonstrate the potential of a novel non-phosphoryl HDAS-PEG for surface modification of preformed liposomes with a goal of prolonging their circulation persistence and more effective inhibition of complement activation.

Synthesis and In Vivo Evaluation of p-18F-Fluorohippurate as a New Radiopharmaceutical for Assessment of Renal Function by PET

The molecular structure of p-18F-fluorohippurate (18F-PFH) is similar to that of p-aminohippurate, a gold standard for the measurement of effective renal plasma flow. The objective of this study was to investigate 18F-PFH as a new PET renal agent. Methods: 18F-PFH was synthesized by reacting N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) with glycine at 90°C (pH 8) for 20 min. In vitro stability was determined by incubating 18F-PFH in fresh human plasma at 37°C for 60 min. In vivo stability was determined by high-performance liquid chromatography analysis of urine collected from a normal rat at 40 min after injection of 18F-PFH. The plasma protein binding and erythrocyte uptake were determined using plasma collected from a normal rat at 5 min after injection of 18F-PFH. The plasma clearance of 18F-PFH was determined using a single-injection clearance method in normal and probenecid-treated rats. Biodistribution studies were conducted in normal rats at 10 min and 1 h after injection of 18F-PFH. Dynamic PET/CT studies were conducted in normal rats injected with 18F-PFH. Results: In normal rats, the plasma clearance of 18F-PFH was 4.11 ± 1.09 mL/min/100 g, which reduced by approximately 50% (P = 0.03) to 2.01 ± 0.08 mL/min/100 g in probenecid-treated rats. About 45.3% of 18F-PFH was found to associate with plasma proteins in vivo in normal rats. Biodistribution studies of 18F-PFH in normal rats showed 72.1 ± 6.4 percentage injected dose and 88.6 ± 6.2 percentage injected dose, respectively, in urine at 10 min and 1 h after injection. The uptake in other organs was negligible. High-performance liquid chromatography analysis of urine collected from a rat at 40 min after injection of 18F-PFH indicated that it was excreted intact, with no metabolic products. Dynamic PET revealed a rapid clearance of 18F-PFH through the renal–urinary pathway. The PET-derived renograms revealed a time to peak activity of 3.0 ± 1.0 min. Conclusion: These combined results warrant further investigation of 18F-PFH as a radiopharmaceutical for the assessment of renal function by PET.

Cyclodextrin-mediated entrapment of curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA in liposomes for treatment of xenograft lung tumor in rats

We recently reported a novel curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA as a potent anti-proliferative agent, and showed that it induces autophagic cell death in lung cancer cells. We are now reporting a drug-in-CD-in-liposome approach to formulate CLEFMA liposomes that could be labeled with Tc-99m radionuclide for non-invasive imaging of their biodistribution. CLEFMA encapsulation was enabled by hydroxypropyl-β-cyclodextrin. In vitro studies showed that CLEFMA possessed more potent anti-proliferative activity in lung adenocarcinoma H441 cells than naturally occurring curcumin. At the same time, it had no effect on the proliferative capacity of normal lung fibroblasts. CLEFMA liposomes retained the antiproliferative potency of free CLEFMA, while maintaining its non-toxic nature in normal lung fibroblasts. In nude rats bearing xenograft H441 tumors, the tumor volume significantly reduced after i.v. treatment with CLEFMA liposomes (p<0.05); the tumor inhibition was determined to be 94%. The anti-tumor activity of CLEFMA liposomes was confirmed by the observation that F-18-fluorodeoxyglucose uptake in tumors of treated rats was reduced as compared to those of control rats. Tc-99m-labeled CLEFMA liposomes accumulated in liver (33.7%); spleen showed the largest accumulation on per gram tissue basis (6.2%/g). Upon histopathological examination of liver, lung and kidney, we found no apparent toxicity from multiple CLEFMA liposome administrations. The results demonstrate the utility of liposomes to serve as a carrier for CLEFMA. This study is the first to demonstrate the efficacy of novel curcuminoid CLEFMA in a preclinical model.

EF24 suppresses maturation and inflammatory response in dendritic cells

Synthetic curcuminoid EF24 was studied for its effect on the maturation and inflammatory response in murine bone marrow derived immortalized JAWS II dendritic cells (DCs). EF24 reduced the expression of LPS-induced MHC class II, CD80 and CD86 molecules. It also abrogated the appearance of dendrites, a typical characteristic of mature DCs. These effects were accompanied by the inhibition of LPS-induced activation of transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). Simultaneous reduction of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, IL-6] both at the mRNA and secreted levels was also observed. To investigate the dependency of LPS effects on MyD88 adaptor protein, we transfected JAWS II DCs with dominant negative MyD88 plasmid construct (MyD88-DN). EF24 reduced NF-κB activity and TNF-α secretion in a MyD88-dependent manner. These results suggest that EF24 modulates DCs by suppressing their maturation and reducing the secretion of inflammatory cytokines. Further, it appears that EF24 acts at or upstream of MyD88 in the LPS-TLR4/MyD88/NF-κB pathway.

Synthesis and Evaluation of Novel Tc-99m Labeled Probestin Conjugates for Imaging APN/CD13 Expression In Vivo

The enzyme aminopeptidase N (APN, also known as CD13) is known to play an important role in tumor proliferation, attachment, angiogenesis, and tumor invasion. In this study, we hypothesized that a radiolabeled high affinity APN inhibitor could be potentially useful for imaging APN expression in vivo. Here, we report synthesis, radiolabeling, and biological evaluation of new probestin conjugates containing a tripeptide, N,N-dimethylglycyl-l-lysinyl-l-cysteinylamide (N(3)S), chelator. New probestin conjugates were synthesized by solid-phase peptide synthesis method, purified by reversed-phase HPLC, and characterized by electrospray mass spectrometry. The conjugates were complexed with Re(V) and (99m)Tc(V) by transmetalation using corresponding Re(V) or (99m)Tc(V) gluconate synthon. The mass spectral analyses of ReO-N(3)S-Probestin conjugates were consistent with the formation of neutral Re(V)O-N(3)S complexes. Initial biological activity of ReO-N(3)S-Probestin conjugates determined by performing an in vitro APN enzyme assay using intact HT-1080 cells demonstrated higher inhibition of APN enzyme activity than bestatin. In vivo biodistribution and whole body planar imaging studies of (99m)TcO-N(3)S-PEG(2)-Probestin performed in nude mice xenografted with human fibrosarcoma tumors derived from HT-1080 cells demonstrated a tumor uptake value of 2.88 ± 0.64%ID/g with tumor-to-blood and tumor-to-muscle ratios of 4.8 and 5.3, respectively, at 1 h postinjection (p.i.). Tumors were clearly visible in whole body planar image obtained at 1 h p.i., but not when the APN was competitively blocked with a coinjection of excess nonradioactive ReO-N(3)S-PEG(2)-Probestin conjugate. These results demonstrate the feasibility of using high affinity APN inhibitor conjugates as targeting vectors for in vivo targeting of APN.

Blast Overpressure Waves Induce Transient Anxiety and Regional Changes in Cerebral Glucose Metabolism and Delayed Hyperarousal in Rats

Physiological alterations, anxiety, and cognitive disorders are strongly associated with blast-induced traumatic brain injury (blast TBI), and are common symptoms in service personnel exposed to blasts. Since 2006, 25,000–30,000 new TBI cases are diagnosed annually in U.S. Service members; increasing evidence confirms that primary blast exposure causes diffuse axonal injury and is often accompanied by altered behavioral outcomes. Behavioral and acute metabolic effects resulting from blast to the head in the absence of thoracic contributions from the periphery were examined, following a single blast wave directed to the head of male Sprague-Dawley rats protected by a lead shield over the torso. An 80 psi head blast produced cognitive deficits that were detected in working memory. Blast TBI rats displayed increased anxiety as determined by elevated plus maze at day 9 post-blast compared to sham rats; blast TBI rats spent significantly more time than the sham controls in the closed arms (p < 0.05; n = 8–11). Interestingly, anxiety symptoms were absent at days 22 and 48 post-blast. Instead, blast TBI rats displayed increased rearing behavior at day 48 post-blast compared to sham rats. Blast TBI rats also exhibited suppressed acoustic startle responses, but similar pre-pulse inhibition at day 15 post-blast compared to sham rats. Acute physiological alterations in cerebral glucose metabolism were determined by positron emission tomography 1 and 9 days post-blast using 18F-fluorodeoxyglucose (18F-FDG). Global glucose uptake in blast TBI rat brains increased at day 1 post-blast (p < 0.05; n = 4–6) and returned to sham levels by day 9. Our results indicate a transient increase in cerebral metabolism following a blast injury. Markers for reactive astrogliosis and neuronal damage were noted by immunoblotting motor cortex tissue from day 10 post-blast in blast TBI rats compared to sham controls (p < 0.05; n = 5–6).

Biological Evaluation of Liposome-Encapsulated Hemoglobin Surface-Modified With a Novel PEGylated Nonphospholipid Amphiphile

Traumatic injury is often associated with hemorrhagic shock. Liposome-encapsulated hemoglobin (LEH) is being developed as an artificial oxygen carrier to address post-hemorrhage oxygen and volume deficit. Here, we report a new composition of LEH based on the use of polyethylene glycol (PEG2K ) conjugated with nonphospholipid hexadecylcarbamoylmethylhexadecanoate (HDAS) to modify the surface of LEH particles. LEH was manufactured by the high-pressure homogenization method using dipalmitoylphosphatidylcholine (∼38 mol%), cholesterol (∼38 mol%), HDAS (∼20 mol%), and highly purified stroma-free human hemoglobin. HDAS-PEG2K was postinserted into the resultant LEH to generate HDAS-PEG2K -LEH. We investigated the potential immune response to HDAS-PEG2K -LEH in a mice model. At the same time, the preparation was tested in a rat model to study the effect of repeated HDAS-PEG2K -LEH injection over 4 weeks. We found that HDAS-PEG2K modification substantially reduced the circulating levels of anaphylatoxins C3a and C5a, as well as plasma levels of thromboxane B2, in mice. Repeated injections of HDAS-PEG2K -LEH in rats did not appear to alter its clearance profile after 4 weeks of treatment. No antibody response against human hemoglobin or PEG was detected in rat plasma. Histological observations of lung, liver, spleen, and kidney were not significantly different between saline-treated rats and HDAS-PEG2K -LEH-treated rats. Immunohistochemical staining for rat heme oxygenase-1 (HO-1) did not show induced expression of HO-1 in these organs. These results suggest that the new surface modification of LEH is immune-neutral and does not adversely affect histology even after repeated administration.