Preclinical and Clinical Use of Indigenously Developed 99mTc-Diethylenetriaminepentaacetic Acid-Bis-Methionine: l-Type Amino Acid Transporter 1-Targeted Single Photon Emission Computed Tomography Radiotracer for Glioma Management.

A new era in tumor classification, diagnosis, and prognostic evaluation has begun as a consequence of recent developments in the molecular and genetic characterization of central nervous system tumors. In this newly emerging era, molecular imaging modalities are essential for preoperative diagnosis, surgical planning, targeted treatment, and post-therapy evaluation of gliomas. The radiotracers are able to identify brain tumors, distinguish between low- and high-grade lesions, confirm a patient's eligibility for theranostics, and assess post-radiation alterations. We previously synthesized and reported the novel l-type amino acid transporter 1 (LAT-1)-targeted amino acid derivative in light of the use of amino acid derivatives in imaging technologies. Further, we have developed a single vial ready to label Tc-lyophilized kit preparations of diethylenetriaminepentaacetic acid-bis-methionine [DTPA-bis(Met)], also referred to as methionine-diethylenetriaminepentaacetic acid-methionine (MDM) and evaluated its imaging potential in numerous clinical studies. This review summarizes our previous publications on 99mTc-DTPA-bis(Met) in different clinical studies such as detection of breast cancer, as a prognostic marker, in detection of recurrent/residual gliomas, for differentiation of recurrent/residual gliomas from radiation necrosis, and for comparison of 99mTc-DTPA-bis(Met) with 11C-L-methionine (11C-MET), with relevant literature on imaging modalities in glioma management.

Sustained Activity of Stimuli-Responsive Curcumin and Acemannan Based Hydrogel Patches in Wound Healing.

Natural plant extract, namely acemannan (Ac) and curcumin (Cur), coencapsulated pluronic micelles, showing thermoresponsive properties, were designed for efficient and safe in vivo wound healing applications. Ac and Cur, widely used antimicrobials, find limited applications because of their low stability, short biological half-life, poor solubility, and low bioavailability. Herein, we report the extraction of Ac from aloe vera and coencapsulation of it with Cur in pluronic micelles to take advantage of the combined effects of both components. Both Ac and Cur preserved their bioactive functionality upon encapsulation. Single photon emission computed tomography imaging confirmed that NPAcC2 hydrogel masked the whole wound by forming a layer. Cur and Ac synergistically resulted in rapid wound closure on the seventh day, and full-grown hair was observed on the 10th day. Individually they both take more than 20 days for wound closure. The increase in the concentration of curcumin increases the healing properties of the material. For days 1, 6, and 10 of the wound dressing experiment, the percentages of wound closure of the mice were the highest for NPAcC2 (i.e., 100%) compared to the untreated control (25%) while maintaining the integrity of the skin. These natural product-based hydrogels have limited side effects vs those caused by commercial drugs in wound healing.

Insights of ligand binding in modeled h5-HT1A receptor: homology modeling, docking, MM-GBSA, screening and molecular dynamics.

The pharmacologically characterized receptor subtype of the serotonin family, the 5HT1A receptor is implicated in the pathophysiology and treatment of depression and anxiety-related disorders. Being the most extensively targeted receptor for developing novel antidepressants and anxiolytics, a near-ideal theoretical model can aid in high-throughput screening of promising drug candidates. However, the design of potential drug candidates suffers owing to a lack of complete structural information. In this work, homology models of 5-HT1A receptor are generated using two distinct alignments (CW and PSTA) and model building methods (KB and EB). The developed models are validated for virtual screening using a ligand dataset of agonists and antagonists. The best-suited model was efficient in discriminating agonist/antagonist binding. Correlation plots between pKi and docking (R2agonist≥ 0.6, R2antagonist≥ 0.7) and MM-GBSA dG bind values (R2agonist≥ 0.5, R2antagonist≥ 0.7) revealed optimum corroboration between in vitro and in silico outcomes, which further suggested the usefulness of the developed model for the design of high-affinity probes for the neurological disorders.Communicated by Ramaswamy H. Sarma.

Quercetin: a savior of alveolar barrier integrity under hypoxic microenvironment.

High altitude pulmonary edema (HAPE) is generally characterized by the loss of alveolar epithelial barrier integrity. The current study was undertaken to assess the noninvasive approaches of HAPE diagnosis and to evaluate the prophylactic potential of quercetin in preventing alveolar junction impairments. Male SD rats fed with quercetin 1 h prior to hypoxia (7,620 m, for 6 h) were selected. PET/CT imaging was performed to visualize the lung uptake of 18F-FDG in animals under hypoxia. Further, oxidant status, catalase activity, hematological & blood gas parameters were evaluated. Moreover, tight junction (TJ) proteins (ZO-1, JAM-C, Claudin-4, and occludin) expression analysis was accomplished using immune-blotting. The structural differences in lung epithelia were noted by TEM imaging. Quercetin prophylaxis has significantly reduced the FDG uptake in rat lungs under hypoxia. It has also dramatically alleviated the protein oxidation followed by an elevation in catalase activity in the lungs under hypoxia. The TJ protein expression in the lungs has also been restored to normal upon quercetin pre-treatment. Concomitantly, the quercetin preconditioning has elicited the stable blood gas and hematological parameters under hypoxia. The observations from TEM imaging have also implicated the normal lung epithelial structures in the quercetin pretreated animals under hypoxia. Quercetin prophylaxis has significantly restored alveolar epithelium integrity by abating oxidative stress in the lungs under hypoxia.Abbreviations: CT- Computed Tomography18F-FDG- Fluorodeoxyglucose (18FHAPE- High Altitude Pulmonary EdemaHb- HemoglobinHCT- HematocritHCO3- BicarbonateJAM- Junctional Adhesion MoleculeKBq- Killo BecquerelPaO2- Partial pressure of arterial oxygenPaCO2- Partial pressure of arterial carbon di-oxidePET- Positron Emission TomographyRBC- Red Blood CorpusclesSD- Sprague DawleyTJ- Tight JunctionsTEM- Transmission Electron MicroscopyWBC- White Blood CorpusclesZO- Zona Occludin.

Biodegradable nanoparticulate co-delivery of flavonoid and doxorubicin: Mechanistic exploration and evaluation of anticancer effect in vitro and in vivo.

The proposed study involves delivering drug/bioactive using a single nanoplatform based on poly lactic-co-glycolic acid (PLGA) for better efficacy, synergistic effect, and reduced toxicity. PLGA was conjugated to doxorubicin (D1), and this conjugate was used for encapsulation of naringenin (D2) to develop naringenin loaded PLGA-doxorubicin nanoparticles (PDNG). The PDNG NPs were 165.4 ± 4.27 nm in size, having 0.112 ± 0.035 PDI, with -10.1 ± 2.74 zeta potential. The surface morphology was confirmed through transmission electron microscopy (TEM) and atomic force microscopy (AFM). The in vitro studies revealed that PDNG NPs exhibited selective anticancer potential in breast cancer cells, and induced apoptosis with S-phase inhibition via an increase in intrinsic reactive oxygen species (ROS) and altering the mitochondrial potential. The results also signified the efficient uptake of nanoparticles encapsulated drugs by cells besides elevating the caspase level suggesting programmed cell death induction upon treatment. In vivo studies results revealed better half-life (27.35 ± 1.58 and 11.98 ± 1.21 h for doxorubicin and naringenin) with higher plasma drug concentration. In vivo biodistribution study was also in accordance with the in vitro studies and in line with the in vivo pharmacokinetic. In vivo tumor regression assay portrayed that the formulation PDNG halts the tumor growth and lessen the tumor volume with the stable bodyweight of the mice. Conclusively, the dual delivery approach was beneficial and highly effective against tumor-induced mice.

Synthesis and biological evaluation of modified laminin peptide (N2S2-KDP) with enhanced affinity for neuronal growth and targeted molecular imaging (SPECT).

An analog of γ1 laminin (RDIAEIIKDI) decapeptide has been used to augment neuronal survival and regeneration after injuries, during aging and other CNS disorder. As a prime synthetic peptide, KDI, is responsible for the neurite outgrowth of human embryonic neurons. In this study, we have designed, modified a KDI derivative and synthesized by replacing isoleucine (I) with Pro (P) amino acid at C-terminal to enhance its potency towards neurite growth. -Cys-Gly-Cys (-CGC) N2S2 motif was also incorporated in the present design for peptide radiolabeling. The modified peptide showed a better binding with the desired 3T1M receptor for neurite growth. The peptide was synthesized using solid phase peptide synthesis and Fmoc-strategy with more than 80% yield. The receptor binding studies of 99mTc-N2S2-KDP in Neuro2A cell lines showed Kd value in 31 nM range and the complex showed appreciable brain uptake in mice. The results on human SH-SY5Y indicate that the unlabeled N2S2-KDP may perhaps be useful for neurite growth in neurodegenerative disorder.

[99mTc]-Bis-Methionine-DTPA Single-Photon Emission Computed Tomography Impacting Glioma Management: A Sensitive Indicator for Postsurgical/Chemoradiotherapy Response Assessment.

Background: The present study evaluated the prognostic value of [99mTc]MDM (bis-methionine-DTPA) follow-up single-photon emission computed tomography (SPECT) imaging for response assessment to chemoradiotherapy in glioma postoperatively. Materials and Methods: One hundred fourteen glioma patients (80 M:34 F) were followed postoperatively by sequential [99mTc]MDM SPECT, dynamic susceptibility contrast-enhanced (DSCE)-MRI, and magnetic resonance spectroscopy (MRS) at baseline, 6, 12, and 22.5 months postchemoradiotherapy. The quantitative imaging results and the clinical outcome were used for response assessment and for the final diagnosis. The quantitative parameter of [99mTc]MDM SPECT were also used for survival analysis. Results: A significantly (p = 0.001) lower target to nontarget (T/NT) ratio was observed in responders than in nonresponders. The sensitivity and specificity of [99mTc]MDM-SPECT for identifying tumor recurrence from radiation necrosis at a cutoff ratio of 1.90 were estimated at 97.9% and 92%. Whereas, the sensitivity and specificity of DSCE-MRI with the normalized cerebral blood volume (nCBV) cutoff of 3.32 for this differentiation was found to be 84.6% and 93.0%. MRS intensity ratios of Cho/NAA and Cho/Cr provided comparatively lower sensitivity of 81.0% and 85.3% and specificity of 73.0% and 73.7%. T/NT ratios correlated with nCBV (r = 0.775, p < 0.001) and to a moderate extent with Cho/NAA ratios (r = 0.467, p = 0.001). [99mTc]MDM SPECT and DSCE-MRI provided comparable results for predicting response assessment to chemoradiotherapy. There was a final diagnosis in 72 patients, of which 47 cases were tumor recurrence and 25 were radiation necrosis. The Kaplan-Meier analysis indicated that patients with T/NT ratio <1.9 showed prolonged survival (53.8 months) as compared (37.2 months) with those who demonstrated T/NT ratio >1.9 (p = 0.0001). Conclusion: Thus, this low-cost SPECT technique in combination with DSCE-MRI can be used accurately for mapping the disease activity, response assessment, and survival in glioma. [99mTc]MDM SPECT and DSCE-MRI had the same diagnostic efficacy to detect recurrent/residual tumor and radiation necrosis while MRS was inferior to both the techniques.

Microenvironment Stimulated Bioresponsive Small Molecule Carriers for Radiopharmaceuticals.

The widespread and successful use of radiopharmaceuticals in diagnosis, treatment, and therapeutic monitoring of cancer and other ailments has spawned significant literature. The transition from untargeted to targeted radiopharmaceuticals reflects the various stages of design and development. Targeted radiopharmaceuticals bind to specific biomarkers, get fixed, and highlight the disease site. A new subset of radioprobes, the bioresponsive radiopharmaceuticals, has been developed in recent years. These probes generally benefit from signal enhancement after undergoing molecular changes due to the fluctuations in the environment (pH, redox, or enzymatic activity) at the site of interest. This review presents a comprehensive overview of bioresponsive radioimaging probes covering the basis, application, and scope of development.

68Ga-Labeled bismacrocyclic methylene phosphonate as potential bone seeking PET radiopharmaceutical.

Phosphonates-based agents are well-known bone-seeking radiopharmaceuticals with application in detection and therapy. With higher sensitivity and resolution offered by Positron Emission Tomography (PET), tracers based on this technique are gaining huge attention. 68Ga-based generator and radiotracers render independence from the on-site cyclotron. We report the development of 68Ga-labeled DOTA-based bismacrocyclic phosphonate derivative, for bone PET imaging. The synthesis and characterization of 68Ga- DO3P-AME-DO3P was carried out in > 95% purity. The radiotracer displayed high stability and low binding affinity (<3%) to blood serum. High in vitro binding affinity were observed for synthetic hydroxyapatite, SAOS-2, osteoclast and osteoblast cells. In vivo pharmacokinetics revealed fast washout with biphasic release pattern. The deposition of radiotracer in osseous tissues was high (Bone/Muscle ratio:18), as studied from the biodistribution studies. In vivo PET/CT and biodistribution analyses revealed the ability of 68Ga-DO3P-AME-DO3P to target and accumulate in bone, thus displaying its potential as a PET bone imaging agent.

The diagnostic performance of 99mTc-methionine single-photon emission tomography in grading glioma preoperatively: a comparison with histopathology and Ki-67 indices.

OBJECTIVE: To characterize glioma preoperatively using quantitative 99mTc-methionine SPECT and comparison with MR-perfusion/spectroscopy and histopatholgical/Ki-67 scoring. METHODS: Twenty-nine patients (21M: 8F; mean age 42.3 ± 10.5 years) with clinical and radiological suspicion of glioma assessed by 99mTc-MDM/SPECT and ceMRI. Additionally, 12/29 patients underwent dynamic susceptibility contrast-enhanced (DSCE) MRI and magnetic resonance spectroscopy (MRS) examination. Three patients with benign pathologies were recruited as controls. Histopathological tumor analysis was done in all (n = 29) the patients, and the Ki-67 index was evaluated in 20/29 patients. The target-to-nontarget (T/NT) methionine tumor uptake ratios, normalized cerebral blood volume (nCBV) and metabolites [choline/N-acetyl aspartate (Cho/NAA), Cho/creatine (Cr), Cr/NAA and Cr/Cho) ratios were measured in tumor areas. RESULTS: On histopathological analysis, 26/29 patients had glioma (G IV-13; G III-04; G II-09). The mean T/NT ratio in G-II was significantly lower (2.46 ± 2.3) than in G-III (7.13 ± 2.2) and G-IV (5.16 ± 1.2). However, the mean ratio was highest (15.9 ± 6.8) in meningioma (n=3). The T/NT cutoff ratio of 3.08 provided 100% sensitivity, 87.5% specificity for discriminating high-grade glioma (HGG) from low-grade glioma (LGG) disease. Likewise, the nCBV cutoff of 2.43 offered 100% sensitivity and 80% specificity. Only the Cho/NAA cutoff value of greater than 3.34 provided reasonable sensitivity and specificity of 85.7% and 80.0% respectively for this differentiation. T/NT ratio correlated significantly with nCBV and Cho/NAA, Cho/Cr ratios but not with Ki-67. CONCLUSION: Quantitative 99mTc-MDM -SPECT provided high sensitivity and specificity to differentiate HGG versus LGG preoperatively and demonstrated a potential role for the differential diagnosis of glial versus nonglial tumors.

Doxorubicin and Crocin Co-delivery by Polymeric Nanoparticles for Enhanced Anticancer Potential In Vitro and In Vivo.

Development of a biodegradable nanoplatform poly(lactic-co-glycolic acid) (PLGA) for co-delivery of two drugs is hugely imperative and beneficial in anticancer therapeutics. In this study, co-delivery of a natural phytoconstituent, crocin (carotenoid), and a commonly prescribed drug, doxorubicin, was attempted using a nanoparticulate platform in the form of PLGA nanoparticles. Doxorubicin was chemically conjugated, while crocin was encapsulated physically in prepared PLGA nanoparticles (PDCR NPs). Prepared NPs were well-characterized for size, ζ, and surface morphology. PDCR NPs were of 174.2 ± 1.57 nm in size. The transmission electron microscopy (TEM) and atomic force microscopy (AFM) images revealed the spherical shape and smooth surface morphology of the nanoparticles, respectively. The entrapment efficiency and drug loading were found to be 58.95 ± 2.58 and 13.89 ± 1.09%, respectively. The drug release pattern of PDCR NPs showed a sustained and controlled release pattern throughout 48 h in PBS buffer pH 7.4 and acetate buffer pH 6.5. PDCR NPs were significantly less hemolytic than doxorubicin (p < 0.0001). Investigational formulation selectively produced cytotoxic effects on breast cancer cells via decreasing reactive oxygen species (ROS) and altering the mitochondrial potential that led to apoptosis with cell-cycle arrest at the G2/M phase. Prepared NPs were able to upregulate the caspase levels as well as efficient uptake by cells in a time-dependent manner. In vivo plasma drug profile studies in healthy rats revealed prolonged persistence of crocin and doxorubicin in systemic circulation. Additionally, the PDCR NPs portrayed reduced tumor volume as compared to control groups in the tumor-induced animal studies, which were favorable. Conclusively, the co-delivery of natural anticancer bioactive crocin along with doxorubicin in PDCR NPs provides a possible controlled-release nanoplatform for efficient drug delivery in vitro and in vivo.

Acetylated Benzothiazolone as Homobivalent SPECT Metallo-Radiopharmaceutical 99mTc-(6-AcBTZ)2DTPA: Design, Synthesis, and Preclinical Evaluation for Mapping 5-HT1A/7 Receptors.

Mapping different structural forms of serotonin subtypes 5-HT1A-5-HT7 using a selective-specific ligand with good pharmacokinetics and brain permeability can open avenues for personalized medication in depressed population. Herein, the selective 5-HT1A/7 antagonist, modified for enhanced brain permeation, is developed as a homobivalent ligand, (6-AcBTZ)2DTPA. After in-depth computational studies to probe the binding mechanism, two-step synthesis lead to (6-AcBTZ)2DTPA. Biocompatibility studies indicated cytocompatibility with 3.6-1.64% cell death (0.1 mM-1 pM) and hemocompatibility with 2.33% hemolysis of human erythrocytes. When 99mTc-radiolabeled in a quantitative yield (98%), a stable preparation was obtained with 7.4 and 3.5% dissociation upon incubation with human serum and excess cysteine. The single-photon-emission computed tomography (SPECT) tracer 99mTc-(6-AcBTZ)2DTPA showed biphasic clearance (t 1/2, distribution = 0.5 min and t 1/2, elimination = 482 min) and maximum brain uptake of 0.42 ± 0.02% ID/g with the regional localization (hippocampus: 11.38% ID/g; cortex: 26.42% ID/g; cerebellum: 25.23% ID/g). Thus, the 99mTc-metal-based SPECT neurotracer holds potential for neuroreceptor mapping.

Evaluation of BBB permeable nucleolipid (NLDPU): A di-C15-ketalised palmitone appended uridine as neuro-tracer for SPECT.

Despite being in routine for onco-diagnostics for years, the applicability of nucleosidic molecular imaging probes is severely restricted in neurological applications due to their low permeability across blood-brain-barrier (BBB). For extending nucleoside tracers utility for neuro-onco early diagnostics, suitable modification which enhances their BBB permeation needs investigation. Among various modifications, lipidization of nucleosides has been reported to enhance cellular permeability. Extending the concept, the aim was to exemplify the possibility of lipidized nucleosides as potential brain tracer with capability to cross intact BBB and evaluate as metal based neuro-imaging SPECT agent. Uridine based non-lipidic (NSDAU) and di-C15-ketal appended lipidic (NLDPU) ligands were conjugated to chelator, DTPA (DTPA-NSDAU and DTPA-NLDPU) using multi-step chemistry. The ligands were evaluated in parallel for comparative physical and biological parameters. Additionally, effects of enhanced lipophilicity on UV-absorption, acid strength, fluorescence and non-specific protein binding were evaluated. Fluorescence quenching of BSA indicated appreciable interaction of DTPA-NLDPU with protein only above 10 mM without inducing conformational changes. In addition, DTPA-NLDPU was found to be haematocompatible and cytocompatible with low dose-dependent toxicity in HEK-cells. The chelator DTPA was used for 99mTc-complexation for SPECT imaging. Optimized 99mTc-radiolabeling parameters resulted in quantitative (≥97%) labeling with good stability parameters in in-vitro serum and cysteine challenge studies. We demonstrate that the nucleolipid radiotracer (99mTc-DTPA-NLDPU) was successfully able to permeate the BBB with brain uptake of 0.2% ID/g in normal mice as compared to 0.06% ID/g uptake of 99mTc-DTPA-NSDAU at 5 min. Blood kinetics indicate biphasic profile and t1/2(distribution) 46 min for 99mTc-DTPA-NLDPU. The preferential accumulation of 99mTc-DTPA-NLDPU in brain tumor intracranial xenograft indicate the targeting capability of the nucleoside. We conclude that as first-of-its-kind, this work presents the potential of the biocompatible nucleolipidic system for brain targeting and early diagnostics.

Optimization by design of etoposide loaded solid lipid nanoparticles for ocular delivery: Characterization, pharmacokinetic and deposition study.

The present study was to develop etoposide loaded solid lipid nanoparticles (SLN) and optimize it for effective ocular delivery to the posterior eye. SLN were prepared by melt-emulsification and ultrasonication technique. Etoposide loaded SLN were optimized by using three-factor three levels Box-Behnken design to establish the functional relationships between variables on responses of particle size, polydispersity index (PDI) and entrapment efficiency (EE). SLN were characterized for size & surface morphology, entrapment efficiency and in vitro release. Further the pharmacokinetic study of optimized formulation after intravitreal administration was evaluated in Wister rats. The deposition in the ocular tissues was checked by scintigraphic analysis in Albino rabbits. Histology was also done to evaluate morphological changes if any occur after treatment. The particle size, PDI and EE obtained for the optimized formulation (Z15) were 239.43 ±â€¯2.35 nm, 0.261 ±â€¯0.001 and 80.96 ±â€¯2.21% respectively. Single intravitreal administrations of SLN were able to give sustained etoposide concentration in the vitreous for 7 consecutive days which was also supported by the results of Gamma scintigraphic study. Histology of posterior ocular tissues do not showed any serious toxic effect. Therefore it can concluded that etoposide loaded SLN was able to maintain vitreous concentration of drug without any serious toxic effect to the surrounding ocular tissues after an intravitreous administration in rat eye.

Radiosynthesis and pre-clinical evaluation of [68Ga] labeled antimicrobial peptide fragment GF-17 as a potential infection imaging PET radiotracer.

The antimicrobial peptide fragment GF-17 was synthesized in-house and conjugated with DOTA and measured molecular mass of DOTA-GF-17 conjugate was 2489 Da. The peptide conjugate was purified and labeled with [68Ga]. The best radiolabeling efficiency (95.0%) of [68Ga]DOTA-GF-17 was achieved at pH 4 with peptide conjugate amount of 20.0 nmol with 30 min of heating at 95 °C. The product remained stable for up to 3 h. The plasma protein binding and lipophilicity for [68Ga]DOTA-GF-17 were 80.98% and -3.12 respectively. The uptake studies with [68Ga]DOTA- GF-17 in S.aureus and P.aeruginosa bacterial strains demonstrated binding of 69.08% and 43.69% respectively. The animal bio-distribution and PET imaging studies were in agreement showing similar pattern for organs' tracer distribution and renal excretion. The tracer had rapid blood clearance and uptake in bone marrow and muscles was very low. The highest uptake of [68Ga]DOTA-GF-17 was observed at 45 min and 120 min in S.aureus and P.aeruginosa infections respectively. [68Ga]DOTA-GF-17 could be a promising PET tracer and holds a great scope for taking the product further to perform extensive PET studies in animal infection (using gram negative/positive strains) models to prove the diagnostic utility of this novel PET tracer for futuristic clinical applications.

Acylated chitosan anchored paclitaxel loaded liposomes: Pharmacokinetic and biodistribution study in Ehrlich ascites tumor bearing mice.

Acylated chitosan (Myristoyl and Octanoyl) coated paclitaxel-loaded liposomal formulation was developed with an aim to overcome the cremophor EL related toxicities. They were evaluated for drug entrapment, in vitro drug release, and cytotoxicity and cell uptake behavior using A549 cells. The 99mTc radio-labeled formulations were also evaluated in vivo in Ehrlich Ascites Tumor (EAT) bearing mice for biodistribution and tumor uptake. The mean particle size of both coated and uncoated liposomal formulations was found to be in the range of 180-200 nm with high drug entrapment efficiency (>90% in case of uncoated liposomes and 80 ±â€¯5% in case of coated liposomes). The uncoated liposomes displayed negative zeta potential (-10.5 ±â€¯4.9 mV) whereas coated liposomes displayed positive zeta potential in the range of +21 to +27 mV. Slower drug release was observed in case of liposomes coated with acylated chitosans as compared to uncoated and native chitosan coated liposomes. All liposomal formulations were found less cytotoxic than paclitaxel injection (Celtax™, Celon Labs, India). In vitro cell uptake and intracellular distribution studies confirmed the cytosolic delivery of uncoated and coated liposomes. The myristoyl chitosan coated liposomal system (LMC) exhibited improved pharmacokinetic, biodistribution and tumor uptake characteristics over other formulations. These obtained results confirmed the potential application of acylated chitosn coated liposomal delivery systems (LMC) in tumor targeting of paclitaxel and other drugs.

Gemini Amphiphile-Based Lipoplexes for Efficient Gene Delivery: Synthesis, Formulation Development, Characterization, Gene Transfection, and Biodistribution Studies.

Some quaternary gemini amphiphiles (GAs) were synthesized as nonviral gene delivery carriers. The critical miceller concentration values of these amphiphiles are indicative of their superior surface-active properties. All of the synthesized GAs, alone or along with lipids like cholesterol and/or dioleoylphosphatidyl ethanolamine (DOPE), were formulated as liposomes. Formulations of GAs with DOPE showed average particle diameters of 326-400 nm with positive ζ-potential (30.1-46.4 mV). The lipoplexes of theses formulations showed complete pDNA retention at the base at a N/P ratio higher than 1.0 in gel retardation study. The GAs were effective in condensing pDNA into a ψ-phase, as indicated by circular dichroism study, and provided complete protection of the pDNA against the enzyme DNase at a N/P ratio more than 1. In vitro cell line studies showed that GA liposomal formulations caused ß-gal expression and offered a higher transfection efficiency than that of liposomes prepared with the help of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP)/DOPE and dicyclocarbodiimide (DCC)/DOPE but comparable to those of Lipofectamine 2000 in A549 and HeLa cell lines. Modulation of head group polarity significantly affected the transfection efficacy of GAs. The cell viabilities of almost all of the formulations were comparable to those of the standards (DCC/DOPE and DOTAP/DOPE liposomes). Incorporation of cholesterol [GA/DOPE/cholesterol in the ratio of 1:1:1] further improved the serum compatibility of the formulations and improved the transfection efficacy when evaluated in A549 and HeLa cell lines. Fluorescence-assisted cell sorting studies showed comparable number of transfected cells to Lipofectamine 2000 in the HeLa cell line. Intracellular trafficking studies using confocal microscopy indicated transfection of the HeLa cells with the reporter gene within 30 min of lipoplex treatment. γ-Scintigraphy using 99mTc-labeled lipoplexes showed higher concentrations of the lipoplexes in vital tissues like liver, spleen, lungs, and kidneys.

Drug-fortified liposomes as carriers for sustained release of NSAIDs: The concept and its validation in the animal model for the treatment of arthritis.

Drug-fortified cationic liposomes of 6­methoxy­2­naphthylacetic acid (6­MNA) were prepared and characterized by various techniques. The residence time of drug-fortified liposomes in joint cavity was evaluated by intra-articular (IA) administration of the radio-labeled (99mTc) liposomal formulation in the inflamed joints in rats. The cationic liposomal formulation composed of 6­MNA (3) as an active agent, its double salt (4) with the lipid 1,2­distearoyl­sn­glycero­3­phosphoethanolamine (DSPE), and pharmaceutically acceptable excipients such as hydrogenated soyabean phospatidylcholine (HSPC) and 1,2­dioleyloxy­3­trimethylammoniumpropane chloride (DOTAP) were developed using thin film hydration technique. The cryo-TEM analysis confirmed that the prepared optimized liposomal formulation (DFL-2) was a mixture of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In addition, the TEM analysis confirmed that the prepared liposomes were of spherical in shape having liposome size in the range of 500-900 nm and zeta potential of about +30 mV. The developed cationic liposomes exhibited sustained release profile of payload of 6­MNA for over >12 h and about five times higher retention in the inflamed animal joints after 24 h (by scintigraphy of the joints) as compared to the plain 6­MNA solution when administered by IA route. The anti-inflammatory activity of prepared liposomal composition is evaluated by Freund's adjuvant induced arthritic model in rats. The liposomal formulation was well tolerated by all animals indicating good biocompatibility. Further, the cationic liposomal formulation treated group showed decreased erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level in comparison to the control and the standard groups in the in vivo study. The improved efficacy of the drug-fortified liposomal formulation was due to the coupled effect of longer retention and sustained release of the active drug 6­MNA in the joints. From the obtained results it could be concluded that the combined effect of the cationic charge on the drug-fortified liposomes and the inherent affinity of the active agent towards the synovial joint tissues, coupled with slow release of the active drug due to double salt approach at the site of administration could potentially decrease the frequency of IA drug administration. Hence such a formulation could prove to be a therapeutic boon for the management of late stage arthritis.