A Comprehensive Review on Exosome: Recent Progress and Outlook.

Exosomes are intrinsic membrane-based vesicles that play a key role in both normal and pathological processes. Since their discovery, exosomes have been investigated as viable drug delivery systems and clinical indicators because of their magnitude and effectiveness in delivering biological components to targeted cells. Exosome characteristics are biocompatible, prefer tumor recruitment, have tunable targeting efficiency, and are stable, making them outstanding and eye-catching medication delivery systems for cancer and other disorders. There is great interest in using cell-released tiny vesicles that activate the immune system in the age of the fast development of cancer immunotherapy. Exosomes, which are cell-derived nanovesicles, have a lot of potential for application in cancer immunotherapy due to their immunogenicity and molecular transfer function. More significantly, exosomes can transfer their cargo to specified cells and so affect the phenotypic and immune-regulation capabilities of those cells. In this article, we summarize exosomes' biogenesis, isolation techniques, drug delivery, applications, and recent clinical updates. The use of exosomes as drug-delivery systems for small compounds, macromolecules, and nucleotides has recently advanced. We have tried to give holistic and exhaustive pieces of information showcasing current progress and clinical updates of exosomes.

Poly-L-lysine Coated Oral Nanoemulsion for Combined Delivery of Insulin and C-Peptide.

An attempt of co-delivery of insulin and C-peptide enclosed in linseed oil globules has been made employing a protective coating of positively charged poly-L-lysine to manage diabetes-associated complications. Oral water in oil in water (w/o/w) nanoemulsion manufactured by double emulsification method showed good entrapment efficiency of 87.6 ± 7.48% for insulin and 73.4 ± 6.44% for C-peptide. The optimized uncoated nanoemulsion showed a mean globule size of 210.6 ± 9.87 nm with a good PDI of 0.145 ± 0.033 and -21.7 ± 4.5 mV ZP. The poly-L-lysine coating of the nanoemulsion resulted in the reversal of surface charge to positive i.e. 18.3 ± 2.7 mV due to the cationic nature of poly-L-lysine. In vitro drug release showed an initial burst of 15-20% release within 4 h followed by controlled release up to 24 h. The poly-L-lysine coated nanoemulsion showed an 8.28-fold higher uptake than fluorescein isothiocyanate (FITC) solution in HCT116 intestinal cell lines. In vivo studies confirmed that orally administered insulin and C-peptide bearing coated nanoemulsion has the potential to improve glycemic control confirmed by blood glucose level under 200 mg/dL for 12 h compared to that of subcutaneous administration of insulin. The formulation was found stable at 25 °C as well as 4°C for up to 3 months. These findings show a promising approach for delivering oral insulin along with C-peptide for effective glycemic control and management of complications associated with diabetes.

Improved chemotherapy against breast cancer through immunotherapeutic activity of fucoidan decorated electrostatically assembled nanoparticles bearing doxorubicin.

Immunotherapeutic nanoparticles (NPs) could be a viable option for delivering cytotoxic agents in a manner which suppresses their toxic manifestations. Doxorubicin (DOX) loaded NPs were prepared using fucoidan (FCD), an immunomodulatory polysaccharide and evaluated against cancer. FCD was electrostatically assembled with cationic polyethylenimine (PEI) through intermolecular electrostatic interactions to develop an immunomodulatory platform to deliver DOX. FCD NPs offered improved cytotoxicity (2.64 folds), cell cycle arrest in G1-S phase (34.65%) and apoptosis (66.12%) in tumor cells compared to free DOX. The enhanced apoptosis was due to raised mitochondrial depolarization (88.00%). In vivo anticancer activity in 4T1 induced tumor bearing BALB/c mice demonstrated a 2.95 folds enhanced efficacy of NPs. Importantly, NPs treatment generated an immunotherapeutic response indicated by gradual increment of the plasma IL-12 levels and reversed polarization of tumor associated macrophages (TAMs) towards M1 subtype. Furthermore, pharmacokinetic study suggested that NPs administration in tumor infested mice caused serum DOX levels to vary in a biphasic pattern, with twin peaks occurring at 1 h and 6 h which help in maintaining preferential drug localization in tumor. Developed NPs would be an excellent approach for improved immune-chemotherapy (in terms of efficacy, safety and immunocompetency) against cancer.

Fabrication of 3-O-sn-Phosphatidyl-L-serine Anchored PLGA Nanoparticle Bearing Amphotericin B for Macrophage Targeting.

PURPOSE: To fabricate, characterize and evaluate 3-O-sn-Phosphatidyl-L-serine (PhoS) anchored PLGA nanoparticles for macrophage targeted therapeutic intervention of VL. MATERIALS AND METHODS: PLGA-AmpB NPs were prepared by well-established nanoprecipitation method and decorated with Phos by thin film hydration method. Physico-chemical characterization of the formulation was done by Zetasizer nano ZS and atomic force microscopy. RESULTS: The optimized formulation (particle size, 157.3 ± 4.64 nm; zeta potential, - 42.51 ± 2.11 mV; encapsulation efficiency, ∼98%) showed initial rapid release up to 8 h followed by sustained release until 72 h. PhoS generated 'eat-me' signal driven augmented macrophage uptake, significant increase in in-vitro (with ∼82% parasite inhibition) and in-vivo antileishmanial activity with preferential accumulation in macrophage rich organs liver and spleen were found. Excellent hemo-compatibility justified safety profile of developed formulation in comparison to commercial formulations. CONCLUSION: The developed PhoS-PLGA-AmpB NPs have improved efficacy, and necessary stability which promisingly put itself as a better alternative to available commercial formulations for optimized treatment of VL.

Chitosan coated PluronicF127 micelles for effective delivery of Amphotericin B in experimental visceral leishmaniasis.

The goal of study was to develop micellar formulation of Amphotericin B (AmB) to improve its antileishmanial efficacy. AmB loaded pluronic F127 (PF 127) micelles were developed and coated with chitosan (Cs-PF-AmB-M) to accord immunoadjuvant and macrophage targeting properties. Hemolysis and cytotoxicity studies demonstrated that Cs-PF-AmB-M was 7.93 fold (at 20µg/ml AmB concentration) and 9.35 fold less hemolytic and cytotoxic, respectively in comparison to AmB suspension. Flow cytometry studies indicated that Cs-PF-FITC-M was 21.97 fold higher internalized byJ774A.1 macrophage in comparison to PF-FITC-M.Cs-PF-AmB-M showed excellent in-vitro (1.82 fold in compared to AmB suspension) and in-vivo (75.84±7.91% parasitic inhibition) antileishmanial activity against macrophage resident intracellular promastigotes and Leishmania donovani infected Syrian hamsters, respectively. Chitosan coating stimulated a Th1 immune response mediating auxiliary immunotherapeutic action as judged by in-vitro and in-vivo cytokine and mRNA expression. Toxicity studies demonstrated normal blood urea nitrogen (BUN) and plasma creatinine (PC) level and no sign of abnormal histopathology upon intravenous administration of micellar formulations. Pharmacokinetic profiling and tissue distribution studies indicated that AmB was preferentially localized in macrophage harboring tissue instead of kidney, thereby circumventing the characteristic nephrotoxicity. Conclusively, Cs-PF-AmB-M could be a viable alternative for the current immuno and chemotherapy of visceral leishmaniasis (VL).

Doxorubicin Hydrochloride Loaded Zymosan-Polyethylenimine Biopolymeric Nanoparticles for Dual 'Chemoimmunotherapeutic' Intervention in Breast Cancer.

OBJECTIVE: To utilize nanoparticles produced by condensation of zymosan (an immunotherapeutic polysaccharide) with pegylated polyethylenimine (PEG-PEI) for dual intervention in breast cancer by modulating tumor microenvironment and direct chemotherapy. METHOD: Positively charged PEG-PEI and negatively charged sulphated zymosan were utilized for electrostatic complexation of chemoimmunotherapeutic nanoparticles (ChiNPs). ChiNPs were loaded with doxorubicin hydrochloride (DOX) for improved delivery at tumor site and were tested for in-vivo tolerability. Biodistribution studies were conducted to showcase their effective accumulation in tumor hypoxic regions where tumor associated macrophages (TAMs) are preferentially recruited. RESULTS: ChiNPs modulated TAMs differentiation resulting in decrement of CD206 positive population. This immunotherapeutic action was furnished by enhanced expression of Th1 specific cytokines. ChiNPs also facilitated an anti-angiogenetic effect which further reduces the possibility of tumor progression and metastasis.

Click Biotinylation of PLGA Template for Biotin Receptor Oriented Delivery of Doxorubicin Hydrochloride in 4T1 Cell-Induced Breast Cancer.

PLGA was functionalized with PEG and biotin using click chemistry to generate a biotin receptor targeted copolymer (biotinylated-PEG-PLGA) which in turn was used to fabricate ultrafine nanoparticles (BPNP) of doxorubicin hydrochloride (DOX) for effective delivery in 4T1 cell induced breast cancer. However, adequate entrapment of a hydrophilic bioactive like DOX in a hydrophobic polymer system made of PLGA is not usually possible. We therefore modified a conventional W/O/W emulsion method by utilizing NH4Cl in the external phase to constrain DOX in dissolved polymer phase by suppressing DOX's inherent aqueous solubility as per common ion effect. This resulted in over 8-fold enhancement in entrapment efficiency of DOX inside BPNP, which otherwise is highly susceptible to leakage due to its relatively high aqueous solubility. TEM and DLS established BPNP to be sized below 100 nm, storage stability studies showed that BPNP were stable for one month at 4 °C, and in vitro release suggested significant control in drug release. Extensive in vitro and in vivo studies were conducted to propound anticancer and antiproliferative activity of BPNP. Plasma and tissue distribution study supplemented by pertinent in vivo fluorescence imaging mapped the exact fate of DOX contained inside BPNP once it was administered intravenously. A comparative safety profile via acute toxicity studies in mice was also generated to out rightly establish usefulness of BPNP. Results suggest that BPNP substantially enhance anticancer activity of DOX while simultaneously mitigating its toxic potential due to altered spatial and temporal presentation of drug and consequently deserve further allometric iteration.

Targeting tumor associated macrophages (TAMs) via nanocarriers.

Recruitment of inflammatory cells to tumor has been well documented, with the most frequent inhabitants being macrophages termed as tumor associated macrophages, (TAMs). Their presence was thought to be an evidence of immune system initiating a fight response towards the tumor, i.e. immune surveillance. This is the case too initially, when TAMs majorly exhibit an M1 phenotype, but their continued presence in tumor microenvironment brings about their polarization to M2 phenotype, which not only participate in continued sustenance of existing tumor but also open up deleterious avenues for further progression and metastasis of cancer. Current perspective is built around this very premise and focuses specifically on TAMs and how they are being targeted by researchers working in annals of nanomedicine. To do so, we dwell into tumor microenvironment and focus on nanotechnology based drug delivery aspects which have either been already or can be potentially employed in the future to target tumor associated macrophages for improved immunoadjuvant therapy of cancer.

Nanoemulsion: Concepts, development and applications in drug delivery.

Nanoemulsions are biphasic dispersion of two immiscible liquids: either water in oil (W/O) or oil in water (O/W) droplets stabilized by an amphiphilic surfactant. These come across as ultrafine dispersions whose differential drug loading; viscoelastic as well as visual properties can cater to a wide range of functionalities including drug delivery. However there is still relatively narrow insight regarding development, manufacturing, fabrication and manipulation of nanoemulsions which primarily stems from the fact that conventional aspects of emulsion formation and stabilization only partially apply to nanoemulsions. This general deficiency sets up the premise for current review. We attempt to explore varying intricacies, excipients, manufacturing techniques and their underlying principles, production conditions, structural dynamics, prevalent destabilization mechanisms, and drug delivery applications of nanoemulsions to spike interest of those contemplating a foray in this field.

Nanosized complexation assemblies housed inside reverse micelles churn out monocytic delivery cores for bendamustine hydrochloride.

OBJECTIVE: We explore a plausible method of targeting bendamustine hydrochloride (BM) to circulatory monocytes by exploiting their intrinsic endocytic/phagocytic capability. METHODS: We do so by complexation of sodium alginate and chitosan inside dioctyl sulfo succinate sodium (AOT) reverse micelles to form bendamustine hydrochloride loaded nanoparticles (CANPs). Dynamic light scattering, electrophoretic mobility and UV spectroscopy were used to detail intra-micellar complexation dynamics and to prove that drug was co-captured during interaction of carbohydrate polymers. A fluorescent conjugate of drug (RBM) was used to trace its intracellular fate after its loading into nanoparticles. RESULTS: CANPs were sized below 150nm, had 75% drug entrapment and negative zeta potential (-30mV). Confocal microscopy demonstrated that developed chitosan alginate nanoparticles had the unique capability to carry BM specifically to its site of action. Quantitative and mechanism based cell uptake studies revealed that monocytes had voracious capacity to internalize CANPs via simultaneous scavenger receptor based endocytic and phagocytic mechanism. Comparative in vitro pharmacokinetic studies revealed obtainment of significantly greater intracellular drug levels when cells were treated with CANPs. This caused reduction in IC50 (22.5±2.1µg/mL), enhancement in G2M cell cycle arrest, greater intracellular reactive oxygen species generation, and increased apopotic potential of bendamustine hydrochloride in THP-1 cells. CONCLUSION: Selective monocytic targeting of bendamustine hydrochloride using carbohydrate constructs can prove advantageous in case of leukemic disorders displaying overabundance of such cells.

Novel Validated RP-HPLC Method for Bendamustine Hydrochloride Based on Ion-pair Chromatography: Application in Determining Infusion Stability and Pharmacokinetics.

Ion pair chromatography was used for quantifying bendamustine hydrochloride (BH) in its marketed vial. The permissive objective was to investigate time duration for which highly susceptible drug content of the marketed vial remained stable after reconstitution. However, the method could also be used to measure extremely low levels of drug in rat plasma and a pharmacokinetic study was accordingly conducted to further showcase method's applicability. Optimized separation was achieved on C-18 Purospher®STAR (250 mm × 4.6 mm, 5 µm particle size) column. Mobile phase flowing at 1.5 mL/min consisted of 5 mM sodium salt of octane sulfonic acid dissolved in methanol, water and glacial acetic acid (55:45:0.075) maintained at pH 6. Detection was carried out at 233 nm with BH eluting after 7.8 min. Validation parameters were determined as per ICH guidelines. Limit of detection and limit of quantification were found to be 0.1 µg/mL and 0.33 µg/mL, respectively. The recoveries were 98-102% in bulk and 85-91% in plasma. The developed method was specific for BH, and utilized for assessing its short-term stability in physiologic solvents and forced degradation products in acid, base, oxidative, light and temperature induced stress environments.

Bridging small interfering RNA with giant therapeutic outcomes using nanometric liposomes.

The scope of RNAi based therapeutics is unquestionable. However, if we dissect the current trend of clinical trials for afore mentioned drug class, some stark trends appear: 1) naked siRNA only exerts influence in topical mode whilst systemic delivery requires a carrier and 2) even after two decades of extensive efforts, not even a single siRNA containing product is commercially available. It was therefore felt that a perspective simplifying the unique intricacies of working with a merger of siRNA and liposomes from a pharmaceutical viewpoint could draw the attention of a wider array of interested researchers. We begin from the beginning and attempt to conduit the gap between theoretical logic and experimental/actual constraints. This, in turn could stimulate the next generation of investigators, gearing them to tackle the conundrum, which is siRNA delivery.