Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100 nm), zeta (–43.3 ± 2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P ≥ 0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2–8°C for more than 3 months. IC50 value of Ambisome (0.18 µg/mL) was comparatively similar to F-1a (0.17 µg/mL) and F-2a (0.16 µg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products

A Cell-Level Systems PK-PD Model to Characterize In Vivo Efficacy of ADCs

Here, we have presented the development of a systems pharmacokinetics-pharmacodynamics (PK-PD) model for antibody-drug conjugates (ADCs), which uses intracellular target occupancy to drive in-vivo efficacy. The model is built based on PK and efficacy data generated using Trastuzumab-Valine-Citrulline-Monomethyl Auristatin E (T-vc-MMAE) ADC in N87 (high-HER2) and GFP-MCF7 (low-HER2) tumor bearing mice. It was observed that plasma PK of all ADC analytes was similar between the two tumor models; however, total trastuzumab, unconjugated MMAE, and total MMAE exposures were >10-fold, ~1.6-fold, and ~1.8-fold higher in N87 tumors. In addition, a prolonged retention of MMAE was observed within the tumors of both the mouse models, suggesting intracellular binding of MMAE to tubulin. A systems PK model, developed by integrating single-cell PK model with tumor distribution model, was able to capture all in vivo PK data reasonably well. Intracellular occupancy of tubulin predicted by the PK model was used to drive the efficacy of ADC using a novel PK-PD model. It was found that the same set of PD parameters was able to capture MMAE induced killing of GFP-MCF7 and N87 cells in vivo. These observations highlight the benefit of adopting a systems approach for ADC and provide a robust and predictive framework for successful clinical translation of ADCs

Covalent functionalized self-assembled lipo-polymerosome bearing Amphotericin B for better management of leishmaniasis and its toxicity evaluation

Amphotericin B remains the preferred choice for leishmanial infection, but it has limited clinical applications due to substantial dose limiting toxicities. In the present work, AmB has been formulated in lipo-polymerosome (L-Psome) by spontaneous self-assembly of synthesized glycol chitosan-stearic acid copolymer. The optimized L-Psome formulation with vesicle size of 243.5 ± 17.9 nm, PDI of 0.168 ± 0.08 and zeta potential of (+) 27.15 ± 0.46 mV with 25.59 ± 0.87% AmB loading was obtained. The field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) images suggest nearly spherical morphology of L-Psome. An in vitro study showed comparatively sustained AmB release (66.082 ± 1.73% within 24 h) and high plasma stability compared to commercial Ambisome and Fungizone, where glycol chitosan content was found to be efficient in preventing L-Psome destabilization in the presence of plasma protein. In vitro and in vivo toxicity studies revealed less toxicity of AmB-L-Psome compared to commercialized Fungizone and Ambisome favored by monomeric form of AmB within L-Psome, observed by UV–visible spectroscopy. Experimental results of in vitro (macrophage amastigote system) and in vivo (Leishmania donovani infected hamsters) illustrated the efficacy of AmB-L-Psome to augment effective antileishmanial properties supported by upregulation of Th-1 cytokines (TNF-α, IL-12 and IFN-γ) and inducible nitric oxide synthase, and downregulation of Th-2 cytokines (TGF-β, IL-10 and IL-4), measured by quantitative mRNA analysis by real time PCR (RT-PCR). Conclusively, developed L-Psome system could be a viable alternative to the current less stable, toxic commercial formulations and developed as a highly efficacious drug delivery system

Self assembled ionically sodium alginate cross-linked amphotericin B encapsulated glycol chitosan stearate nanoparticles: applicability in better chemotherapy and non-toxic delivery in visceral leishmaniasis

Objectives: To investigate the applicability, localization, biodistribution and toxicity of self assembled ionically sodium alginate cross-linked AmB loaded glycol chitosan stearate nanoparticles for effective management of visceral leishmaniasis. Methods: Here, we fabricated Amphotericin B (AmB) encapsulated sodium alginate-glycol chitosan stearate nanoparticles (AmB-SA-GCS-NP) using strong electrostatic interaction between oppositely charged polymer and copolymer by ionotropic complexation method. The tagged FAmB-SA-GCS-NP was compared with tagged FAmB for in vitro macrophagic uptake in J774A macrophages and in vivo localization in liver, spleen, lung and kidney tissues. The AmB-SA-GCS-NP and plain AmB were compared for in vitro and in vivo antileishmanial activity, pharmacokinetics, organ distribution and toxicity profiling. Results: The morphology of SA-GCS-NP revealed as nanocrystal (size, 196.3 ± 17.2 nm; PDI, 0.216 ± 0.078; zeta potential, (−) 32.4 ± 5.1 mV) by field emission scanning electron microscopy and high resolution transmission electron microscopy. The macrophage uptake and in vivo tissue localization studies shows tagged FAmB-SA-GCS-NP has significantly higher (~1.7) uptake compared to tagged FAmB. The biodistribution study of AmB-SA-GCS-NP showed more localized distribution towards Leishmania infected organs i.e. spleen and liver while lesser towards kidney. The in vitro (IC<SUB>50</SUB>, 0.128 ± 0.024 μg AmB/ml) and in vivo (parasite inhibition, 70.21 ± 3.46%) results of AmB-SA-GCS-NP illustrated significantly higher (P &#60; 0.05) efficacy over plain AmB. The monomeric form of AmB within SA-GCS-NP, observed by UV-visible spectroscopy, favored very less in vitro and in vivo toxicities compared to plain AmB. Conclusion: The molecular organization, toxicity studies, desired localization and biodistribution of cost effective AmB-SA-GCS-NP was found to be highly effective and can be proved as practical delivery platform for better management of leishmaniasis

Vitamin B₁₂ Grafted Layer-by-Layer Liposomes Bearing <i>HBsAg</i> Facilitate Oral Immunization: Effect of Modulated Biomechanical Properties

Adhesion forces of nanoparticulate materials toward biological membrane are crucial for designing a delivery system for therapeutic molecules and vaccines. The present study aims to investigate the impact of surface roughness of the nanoparticulate system in oral delivery of antigen and its targeting to toward intestinal antigen presenting cells. To evaluate this hypothesis, layer-by-layer coated liposomes (LBL-Lipo) were fabricated using sodium alginate and Vitamin B₁₂ conjugated Chitosan (VitB₁₂–Chi) as anionic and cationic polyelectrolyte, respectively. Change in surface roughness was observed on changes in pH from gastric to intestinal conditions attributed to increase and decrease in charge density on VitB₁₂–Chi. Surface roughness was measured in terms of root–mean–square measured by topographical analysis using atomic force microscopy. LBL-Lipo were further characterized for their size, zeta potential, and release behavior to evaluate the potential for oral vaccine delivery. In vitro cell uptake in macrophage cells (J-744) shows about 2- and 3.1-fold increased uptake of rough LBL-Lipo over smooth LBL-Lipo at 37 °C (endocytosis) and 4 °C (endocytosis inhibition) indicating improved biological interaction. Further in vivo immunization study revealed that prototype formulations were able to produce 4.8- and 3.3-fold higher IgG and IgA levels in serum and feces, respectively, in comparison to smooth LBL-Lipo

Covalent Functionalized Self-Assembled Lipo-Polymerosome Bearing Amphotericin B for Better Management of Leishmaniasis and Its Toxicity Evaluation

Amphotericin B remains the preferred choice for leishmanial infection, but it has limited clinical applications due to substantial dose limiting toxicities. In the present work, AmB has been formulated in lipo-polymerosome (L-Psome) by spontaneous self-assembly of synthesized glycol chitosan-stearic acid copolymer. The optimized L-Psome formulation with vesicle size of 243.5 ± 17.9 nm, PDI of 0.168 ± 0.08 and zeta potential of (+) 27.15 ± 0.46 mV with 25.59 ± 0.87% AmB loading was obtained. The field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) images suggest nearly spherical morphology of L-Psome. An <i>in vitro</i> study showed comparatively sustained AmB release (66.082 ± 1.73% within 24 h) and high plasma stability compared to commercial Ambisome and Fungizone, where glycol chitosan content was found to be efficient in preventing L-Psome destabilization in the presence of plasma protein. <i>In vitro</i> and <i>in vivo</i> toxicity studies revealed less toxicity of AmB-L-Psome compared to commercialized Fungizone and Ambisome favored by monomeric form of AmB within L-Psome, observed by UV–visible spectroscopy. Experimental results of <i>in vitro</i> (macrophage amastigote system) and <i>in vivo</i> (<i>Leishmania donovani</i> infected hamsters) illustrated the efficacy of AmB-L-Psome to augment effective antileishmanial properties supported by upregulation of Th-1 cytokines (TNF-α, IL-12 and IFN-γ) and inducible nitric oxide synthase, and downregulation of Th-2 cytokines (TGF-β, IL-10 and IL-4), measured by quantitative mRNA analysis by real time PCR (RT-PCR). Conclusively, developed L-Psome system could be a viable alternative to the current less stable, toxic commercial formulations and developed as a highly efficacious drug delivery system

New Fluoranthene FLUN-550 as a Fluorescent Probe for Selective Staining and Quantification of Intracellular Lipid Droplets

A new class of live cell permeant, nontoxic fluoranthene-based fluorescent probe (FLUN-550) having a high Stokes shift in aqueous medium has been discovered. It showed selective staining of lipid droplets (LDs, dynamic cytoplasmic organelles) at a low concentration without background noise in in vitro live cell imaging of 3T3-L1 preadipocytes, J774 macrophages, MCF7 breast cancer cells, and single-celled, parasitic protozoa <i>Leishmania donovani</i> promastigotes and in vivo nonparasitic soil nematode <i>C. elegans</i>

Development of docetaxel nanocapsules for improving <i>in vitro</i> cytotoxicity and cellular uptake in MCF-7 cells

<p>The aim of this study was to fabricate docetaxel loaded nanocapsules (DTX-NCs) with a high payload using Layer-by-Layer (LbL) technique by successive coating with alternate layers of oppositely charged polyelectrolytes. Developed nanocapsules (NCs) were characterized in terms of morphology, particle size distribution, zeta potential (ζ-potential), entrapment efficiency and <i>in vitro</i> release. The morphological characteristics of the NCs were assessed using transmission electron microscopy (TEM) that revealed coating of polyelectrolytes around the surface of particles. The developed NCs successfully attained a submicron particle size while the ζ-potential of optimized NCs alternated between (+) 34.64 ± 1.5 mV to (−) 33.25 ± 2.1 mV with each coating step. The non-hemolytic potential of the NCs indicated the suitability of the developed formulation for intravenous administration. A comparative study indicated that the cytotoxicity of positively charged NCs (F4) was significant higher (<i>p</i> < 0.05) rather than negative charged NCs (F3), plain drug (DTX) and marketed preparation (Taxotere®) when evaluated <i>in vitro</i> on MCF-7 cells. Furthermore, cell uptake studies evidenced a higher uptake of positive NCs (≥1.2 fold) in comparison to negative NCs. In conclusion, formulated NCs are an ideal vehicle for passive targeting of drugs to tumor cells that may result in improved efficacy and reduced toxicity of encapsulated drug moiety.</p