Sterically Stabilized Micelles Research Articles

Targeting breast cancer using pirarubicin-loaded vasoactive intestinal peptide grafted sterically stabilized micelles

In this study the chemotherapeutic agent Pirarubicin (PRB) which is known for its serious side effects was actively targeted to the breast cancer cells by uploading it to the biocompatible and biodegradable Sterically Stabilized Micelles (SSMs) made of 1,2- Distearoyl- sn- glycero‑3- phosphoethanolamine- N- methoxy‑ polyethylene glycol 2000 (DSPE-PEG2000) to enhance efficacy and reduce toxicity. Vasoactive intestinal peptide (VIP), the receptors of which are overexpressed on the breast cancer cells, was grafted on the surface of the micelles. To the best of our knowledge this is the first report on active targeting of PRB to tumor site.For this purpose, PRB loaded VIP grafted SSMs (PRB-SSM-VIP) were synthesized and characterized. The in vitro efficiency of PRB-SSM-VIP along with SSM and free PRB was investigated on the MCF-7 breast cancer cells and the in vivo effects were studied on the 4T1 breast cancer bearing nude mice.Solubilizing 300 µg of PRB using 2.81 mg of DSPE-PEG2000 resulted in obtaining monodispersed particles of 12.16 ± 2.7 nm with slow drug release profile. Incorporation of PRB within the hydrophobic DSPE core of SSM was confirmed using differential scanning calorimetry (DSC) and the spherical shape of the synthesized particles was demonstrated using atomic force microscope (AFM).Both in vitro and in vivo studies showed significantly higher activity of PRB-SSM-VIP compared to free PRB. In vivo imaging showed successful accumulation of PRB-SSM-VIP at the tumor site and 98.8% tumor eradication was obtained with no signs of side effects. Current study suggests that SSM-VIP could be used as new drug delivery system for targeting PRB to the breast cancer cells.

Development of co-solvent freeze-drying method for the encapsulation of water-insoluble thiostrepton in sterically stabilized micelles

The purpose of this work was to develop a practical and scalable method to encapsulate the hydrophobic antibiotic thiostrepton (TST) in sterically stabilized micelles (SSM). Using the conventional method of thin-film hydration, we encapsulated up to 5 drug molecules per SSM (diameter ∼ 16 nm). However, since this method is not suitable for large-scale production – a limiting factor for clinical translation – we applied the co-solvent freeze-drying method using tert-butanol (TBA): water co-solvent system. We found that the presence of phosphate-buffered saline (PBS) salts in the lyophilized cake accelerated the reconstitution time and allowed efficient drug encapsulation without the formation of larger drug particles. In addition, TBA proportion of 50% (v/v) was sufficient to maintain both phospholipid and drug in solution prior to the freeze-drying. The increase of drug and phospholipid concentrations in the formulation extended the reconstitution time and led to drug precipitation. Therefore, to increase the strength of the formulation, we prepared lyophilized cakes with lower phospholipid content (5 mM) and reconstituted them in one-third of the fill volume. In conclusion, we found optimum conditions to prepare TST-SSM using the co-solvent freeze-drying method. This scalable production method can facilitate the further clinical development and industrial production of TST-SSM nanomedicine.

Cytotoxic, Apoptotic and Genotoxic Effects of Lipid-Based and Polymeric Nano Micelles, an In Vitro Evaluation.

Self-assembly systems (SAS) mainly consist of micelles, and liposomes are the classes of Nano Drug Delivery Systems with superior properties compared to traditional therapeutics in targeting cancer tumors. All commercially available nano-formulations of chemotherapeutics currently consist of SAS. According to our knowledge, a specific toxicity comparison based on material differences has not yet been performed. The purpose of this study was to evaluate and compare the toxicity of two SAS consisting of Sterically Stabilized Micelles (SSM) made of a lipid-based amphiphilic distearoyl-sn-glycero-phosphatidylethanolamine-polyethylene glycol (PEG)-2000 and a polymeric micelle (PM) consisting of Y-shape amphiphilic block copolymer, synthesized using poly ε-caprolactone and PEG. The mechanism of cytotoxicity and genotoxicity of micelles on L-929 healthy mouse fibroblast cells was assessed using Sulforhodamine-B, WST-1, Acridine Orange/Ethidium Bromide and alkaline single-cell gel electrophoresis assays. Results showed that SSM in conc. of 40 mg/mL shows very low cytotoxicity at the end of 24, 48 and 72 h. The DNA damage caused by SSM was much lower than PM while the latter one showed significant toxicity by causing apoptosis with the ED50 value of 3 mg/mL. While the DNA damage caused by SSM was ignorable, some DNA chain breaks were detected on cells treated with PM.

Micellar Nanomedicine of Novel Fatty Acid Modified Xenopus Glucagon-like Peptide-1: Improved Physicochemical Characteristics and Therapeutic Utilities for Type 2 Diabetes.

To develop novel long-acting antidiabetics with improved therapeutic efficacy, two glucagon-like peptide-1 (GLP-1) analogs were constructed through the hybridization of key sequences of GLP-1, xenGLP-1B, exendin-4, and lixisenatide. Hybrids 1 and 2 demonstrated enhanced in vitro and in vivo biological activities and were further site-specifically lipidized at lysine residues to achieve prolonged duration of action and less frequent administration. Compared with their native peptides, compounds 3-6 showed similar in vitro activities but impaired in vivo acute hypoglycemic potencies due to decreased aqueous solubility and retarded absorption in vivo. To circumvent these issues, compound 3 (xenoglutide) was selected to be self-associated with sterically stabilized micelles (SSM). The α-helix and solubility of xenoglutide were significantly improved after self-associated with SSM. Notably, the improved physicochemical characteristics of xenoglutide-SSM led to revival of acute hypoglycemic ability without affecting its long-term glucose-lowering activity. Most importantly, preclinical studies demonstrated improved therapeutic effects and safety of xenoglutide-SSM in diabetic db/db mice. Our work suggests the SSM incorporation as an effective approach to improve the pharmacokinetic and biological properties of hydrophobicity peptide drugs. Furthermore, our data clearly indicate xenoglutide-SSM as a novel nanomedicine for the treatment of type 2 diabetics.

An approach for liposome immobilization using sterically stabilized micelles (SSMs) as a precursor for bio-layer interferometry-based interaction studies

Non-fluidic bio-layer interferometry (BLI) has rapidly become a standard tool for monitoring almost all biomolecular interactions in a label-free, real-time and high-throughput manner. High-efficiency screening methods which measure the kinetics of liposomes with a variety of compounds require the immobilization of liposomes. In this work, a method is described for immobilizing liposomes for interaction studies, based on the biophysical principles of this biosensor platform. The immobilization approach includes the loading of DSPE-PEG(2000)-biotin containing sterically stabilized micelles (SSMs) which are restructured in a buffer change step, resulting in an accessible substrate for liposome immobilization. Liposomes in a concentration of 5mM of varying composition and fluidity were immobilized on the sensor surface by inserting the hydrophobic residues of the former loaded SSMs. This proof of principle was carried out using Cytochrome C as a membrane-interacting model protein. The binding of Cytochrome C to the immobilized liposomes was demonstrated, and the derived kinetic and affinity constants were similar to values given in the literature. In order to obtain a detailed understanding of this surface, and to show the integrity of the liposomes, confocal fluorescence microscopy was used. Images of immobilized liposomes containing calcein in the aqueous core indicated intact vesicles. A combination of this simple liposome immobilization approach, the possibility of automation on BLI systems with high throughput within an acceptable timescale and excellent reproducibility makes this assay suitable for basic research as well as for industrial and regulatory applications.

Target selective micelles for bombesin receptors incorporating Au(III)-dithiocarbamato complexes

Pure sterically stabilized micelles (SSM) of DSPE-PEG2000, and sterically stabilized mixed micelles (SSMM) containing PC or DOPC phospholipids (5, 10 or 20% mol/mol with respect to DSPE-PEG2000) are developed as delivery systems for the gold based cytotoxic drug Au(III)-dithiocarbamato complex AuL12. In particular, SSMM containing 5% of PC at 5mM of lipid concentration encapsulates 61.0μg of AuL12 with a DL% of 1.13. The gold complex remains stable up to 72h when incorporated in the aggregate, as indicated by UV–vis measurements. Incorporation in micelle composition of a low amount of the peptide derivative MonY-BN-AA1, containing a bombesin peptide analogue does not influence structural parameters of the micelles (diameter around 20nm) neither the AuL12 loading parameters. Target selective properties of the peptide containing full aggregate on PC-3 cells overexpressing the GRP/bombesin receptors are observed by in vitro cytotoxic studies: a decrease of cell viability, ∼50%, is obtained in cells treated with AuL12-targeted micelles at 10μM drug concentration for 48h with respect to untargeted micelles.

Novel, Biocompatible, and Disease Modifying VIP Nanomedicine for Rheumatoid Arthritis

Despite advances in rheumatoid arthritis (RA) treatment, efficacious and safe disease-modifying therapy still represents an unmet medical need. Here, we describe an innovative strategy to treat RA by targeting low doses of vasoactive intestinal peptide (VIP) self-associated with sterically stabilized micelles (SSMs). This spontaneous interaction of VIP with SSM protects the peptide from degradation or inactivation in biological fluids and prolongs circulation half-life. Treatment with targeted low doses of nanosized SSM-VIP but not free VIP in buffer significantly reduced the incidence and severity of arthritis in an experimental model, completely abrogating joint swelling and destruction of cartilage and bone. In addition, SSM associated VIP, unlike free VIP, had no side-effects on the systemic functions due to selective targeting to inflamed joints. Finally, low doses of VIP in SSM successfully downregulated both inflammatory and autoimmune components of RA. Collectively, our data clearly indicate that VIP-SSM should be developed to be used as a novel nanomedicine for the treatment of RA.

A novel peptide nanomedicine for treatment of pancreatogenic diabetes

Pancreatogenic diabetes (PD) is a potentially fatal disease that occurs secondary to pancreatic disorders. The current anti-diabetic therapy for PD is fraught with adverse effects that can increase morbidity. Here we investigated the efficacy of novel peptide nanomedicine: pancreatic polypeptide (PP) in sterically stabilized micelles (SSM) for management of PD. PP exhibits significant anti-diabetic efficacy but its short plasma half-life curtails its therapeutic application. To prolong and improve activity of PP in vivo, we evaluated the delivery of PP in SSM. PP-SSM administered to rats with PD, significantly improved glucose tolerance, insulin sensitivity and hepatic glycogen content compared to peptide in buffer. The studies established the importance of micellar nanocarriers in protecting enzyme-labile peptides in vivo and delivering them to target site, thereby enhancing their therapeutic efficacy. In summary, this study demonstrated that PP-SSM is a promising novel anti-diabetic nanomedicine and therefore should be further developed for management of PD. From the Clinical EditorPancreatic peptide was earlier demonstrated to address pancreatogenic diabetes, but its short half-life represented major difficulties in further development for therapeutic use. PP-SSM (pancreatic polypeptide in sterically stabilized micelles) is a promising novel anti-diabetic nanomedicine that enables prolonged half-life and increased bioactivity of PP, as shown in this novel study, paving the way toward clinical studies in the near future.

Human Pancreatic Polypeptide in a Phospholipid-Based Micellar Formulation

Pancreatic polypeptide (PP) has important glucoregulatory functions and thereby holds significance in the treatment of diabetes and obesity. However, short plasma half-life and aggregation propensity of PP in aqueous solution, limits its therapeutic application. To address these issues, we prepared and characterized a formulation of PP in sterically stabilized micelles (SSM) that protects and stabilizes PP in its active conformation. PP-SSM was prepared by incubating PP with SSM dispersion in buffer. Peptide-micelle association and freeze-drying efficacy of the formulation was characterized in phosphate buffers with or without sodium chloride using dynamic light scattering, fluorescence spectroscopy and circular dichroism. The degradation kinetics of PP-SSM in presence of proteolytic enzyme was determined using HPLC and bioactivity of the formulation was evaluated by in vitro cAMP inhibition study. PP self-associated with SSM and this interaction was influenced by presence/absence of sodium chloride in the buffer. The formulation was effectively lyophilized, demonstrating feasibility for its long-term storage. The stability of peptide against proteolytic degradation was significantly improved and PP in SSM retained its bioactivity in vitro. Self-association of PP with phospholipid micelles addressed the delivery issues of the peptide. This nanomedicine should be further developed for the treatment of diabetes.

Actively Targeted Low-Dose Camptothecin as a Safe, Long-Acting, Disease-Modifying Nanomedicine for Rheumatoid Arthritis

Camptothecin (CPT), a potent topoisomerase I inhibitor, was originally discovered as an anticancer agent to induce programmed cell death of cancer cells. Recent evidence suggests that, similar to cancer, alterations in apoptosis and over-proliferation of key effector cells in the arthritic joint result in rheumatoid arthritis (RA) pathogenesis. Initial in vitro studies have suggested that camptothecin inhibits synoviocyte proliferation, matrix metalloproteinases expression in chrondrocytes and angiogenesis. This study is one of the first to test, in vivo, RA as a new indication for CPT. To circumvent insolubility, instability and toxicity of CPT, we used biocompatible, biodegradable and targeted sterically stabilized micelles (SSM) as nanocarriers for CPT (CPT-SSM). We also surface-modified CPT-SSM with vasoactive intestinal peptide (VIP) for active targeting. We then determined whether this nanomedicine abrogated collagen-induced arthritis (CIA) in mice. Based on our findings, this is the first study to report that CPT was found to be efficacious against CIA at concentrations significantly lower than usual anti-cancer dose. Furthermore, a single subcutaneous injection of CPT-SSM-VIP (0.1 mg/kg) administered to CIA mice mitigated joint inflammation for at least 32 days thereafter without systemic toxicity. CPT alone needed at least 10-fold higher dose to achieve the same effect, albeit with some vacuolization in liver histology. We propose that CPT-SSM-VIP is a promising targeted nanomedicine and should be further developed as a safe, long-acting, disease-modifying pharmaceutical product for RA.

Self-associated indisulam in phospholipid-based nanomicelles: a potential nanomedicine for cancer

This study aimed to begin development of a nanomedicine containing indisulam solubilized in sterically stabilized micelles (SSMs) composed of DSPE-PEG 2000 or sterically stabilized mixed micelles (SSMMs) composed of DSPE-PEG 2000 plus egg phosphatidylcholine. Micelles were prepared by co-precipitation and reconstitution of drug and lipids. Particle size distributions of micellar formulations were determined by quasi-elastic light scattering. Amounts of solubilized drug were determined by reverse-phase high-performance liquid chromatography (RP-HPLC). In vitro cytotoxicity of indisulam in nanocarrier was determined on the MCF-7 cell line by the National Cancer Institute–developed sulforhodamine B assay. Optimal solubilized indisulam concentrations in 5 mM total lipid were 10 μg/mL for SSMMs and 400 μg/mL for SSMs. HPLC results demonstrated that the encapsulation capacity of both micelles was over 95%. In vitro studies showed that indisulam in micellar system was more effective than free indisulam. The optimized formulation was successfully freeze-dried without any addition of lyoprotectants or cryoprotectants. We conclude that SSMs are a promising nanocarrier for indisulam, and indisulam-SSMs should be developed further as a novel targeted nanomedicine.

Camptothecin in sterically stabilized phospholipid micelles: A novel nanomedicine

Background Camptothecin (CPT) is a well-established topoisomerase I inhibitor against a broad spectrum of cancers. However, poor aqueous solubility, instability, and toxic effects to normal tissues have limited CPT clinical development. Recently, sterically stabilized micelles (SSM) composed of polyethylene glycol (PEGylated) phospholipids have been introduced as safe, biocompatible nanocarriers for the delivery of poorly water-soluble drugs. It was the aim of this study to develop and evaluate in vitro camptothecin-containing SSM (CPT-SSM) as a novel nanomedicine for parenteral administration. Methods The solubilization potential, stability, and in vitro cytotoxicity of CPT in SSM were studied. Lyophilization of CPT-SSM under controlled conditions was also studied. Results The mean size of CPT-SSM was found to be ∼14 nm with a narrow size distribution. CPT-SSM were prepared by coprecipitation reconstitution. At a concentration of 15 mmol/L of PEGylated phospholipids where no micelle–micelle interaction was observed, CPT solubilization in SSM was 25-fold higher than CPT in buffer. We determined that CPT can be solubilized in SSM up to molar ratios of CPT/lipid = 0.0063:1. Above this critical molar ratio, heterogeneous systems of CPT-SSM and CPT self-aggregated particles were formed. CPT in SSM was at least 3 times more stable and 3-fold more cytotoxic to MCF-7 cells than CPT alone. Furthermore, CPT-SSM alone was lyophilized without additional lyoprotectants and cryoprotectants and reconstituted without any significant change in properties. Conclusion We have shown that CPT in SSM is a promising nanomedicine with improved drug solubility, stability, freeze-drying properties, and anticancer activity. It is anticipated that, because of the nanosize and steric stability of the micelles, CPT-SSM will be passively targeted to solid cancers in vivo, resulting in high drug concentration in tumors and reduced drug toxicity to the normal tissues.

In vitro characterization of PEGylated phospholipid micelles for improved drug solubilization: Effects of PEG chain length and PC incorporation

Sterically stabilized micelles (SSM) composed of poly(ethylene glycol-2000)-grafted distearoylphosphatidylethanolamine (DSPE-PEG) and sterically stabilized mixed micelles (SSMM) composed of DSPE-PEG and egg-phosphatidyl choline (PC) have recently been introduced as novel lipid based carriers for water-insoluble drugs. However, factors that affect the solubilization behavior of these phospholipid micelles are not well understood. This study investigates the effect of PEG chain length and PC content on physical properties and solubilization potential of PEGylated phospholipid micelles. Critical micelle concentrations (CMC) determined for DSPE-PEG with different PEG chain lengths (2000, 3000, and 5000) using a fluorescent probe were in the micromolar range (0.5−1.5μM) with higher CMC for longer PEG chain length. The size of micelles determined by quasi-elastic light scattering (QELS) showed that micellar systems became heterogeneous when PC was added at ≥25% for DSPE-PEG 2000 and≥40% for DSPE-PEG 5000, respectively. Above these critical PC ratios a significant increase in aggregation number and formation of rodlike particles were observed by small angle neutron scattering (SANS). Solubilization of diazepam was greater with DSPE-PEG 2000 than DSPE-PEG 5000 simple micelles as determined by RP-HPLC. However, DSPE-PEG 5000 micelles showed greater improvement in solubilization of the water-insoluble drug with an increase in PC content. In conclusion, phospholipid micelle size and solubilization potential varied with PEG chain length and PC content in the mixed micelle. Aggregation number and shape of the micelles did not significantly change until the critical PC concentrations.

Effects of peptide molecular mass and PEG chain length on the vasoreactivity of VIP and PACAP 1–38 in pegylated phospholipid micelles

Bioactive properties of certain amphipathic peptides are amplified when self-associated with sterically stabilized micelles (SSM) composed of polyethylene glycol (PEG)-conjugated phospholipids. The purpose of this study was to determine the effects of amphipathic peptide molecular mass and PEG chain length on vasoreactivity evoked by vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, and pituitary adenylate cyclase-activating peptide 1–38 (PACAP 1–38), a 38-amino acid neuropeptide, associated with PEGylated phospholipid micelles in vivo. Both peptides were incubated for 2 h with SSM composed of PEG with molecular mass of 2000 or 5000 grafted onto distearoyl-phosphatidylethanolamine (DSPE-PEG2000 or DSPE-PEG5000) before use. We found that regardless of peptide molecular mass, PEG chain length had no significant effects on peptide–SSM interactions. Using intravital microscopy, VIP associated with DSPE-PEG5000 SSM or DSPE-PEG2000 SSM incubated at 25 °C evoked similar vasodilation in the intact hamster cheek pouch microcirculation. Likewise, PACAP 1–38-induced vasodilation was PEG chain length-independent. However, SSM-associated PACAP 1–38 evoked significantly smaller vasodilation than that evoked by SSM-associated VIP ( P < 0.05) at 25 °C. When the incubation temperature was increased to 37 °C, SSM-associated PACAP 1–38-induced vasodilation was now similar to that of SSM-associated VIP. This response was associated with a corresponding increase in α-helix content of both peptides in the presence of phospholipids. Collectively, these data indicate that for a larger amphipathic peptide, such as PACAP 1–38, greater kinetic energy or longer incubation period is required to optimize peptide–SSM interactions and amplify peptide bioactivity in vivo.