Drug Release Patterns and Cytotoxicity of PEG-poly(aspartate) Block Copolymer Micelles in Cancer Cells

Purpose

To test physicochemical and biological properties of PEG-poly(aspartate) [PEG-p(Asp)] block copolymer micelles entrapping doxorubicin hydrochloride (DOX) through ionic interaction.

Methods

PEG-p(Asp) was synthesized from 5?kDa PEG and 20 Asp units. Carboxyl groups of p(Asp) were present as benzyl ester [PEG-p(Asp/Bz)], sodium salt [PEG-p(Asp/Na)] or free acid [PEG-p(Asp/H)]. Block copolymers and DOX were mixed at various ratios to prepare polymer micelles, which were subsequently characterized to determine particle size, drug loading and release patterns, and cytotoxicity against prostate (PC3 and DU145) and lung (A549) cancer cell lines.

Results

PEG-p(Asp/Bz), Na- and H-micelles entrapped 1.1, 56.8 and 40.6?wt.% of DOX, respectively. Na- and H-micelles (<100?nm) showed time-dependent DOX release at pH 7.4, which was accelerated at pH 5.0. Na-micelles were most stable at pH 7.4, retaining 31.8% of initial DOX for 48?h. Cytotoxicity of Na-micelles was 23.2% (A549), 28.5% (PC3) and 45.9% (DU145) more effective than free DOX.

Conclusion

Ionic interaction appeared to entrap DOX efficiently in polymer micelles from PEG-p(Asp) block copolymers. Polymer micelles possessing counter ions (Na) of DOX in the core were the most stable, releasing drugs for prolonged time in a pH-dependent manner, and suppressing cancer cells effectively.     

Polyplex Micelles from Triblock Copolymers Composed of Tandemly Aligned Segments with Biocompatible, Endosomal Escaping, and DNA-Condensing Functions for Systemic Gene Delivery to Pancreatic Tumor Tissue

Purpose  For systemic gene delivery to pancreatic tumor tissues, we prepared a three-layered polyplex micelle equipped with biocompatibility, efficient endosomal escape, and pDNA condensation functions from three components tandemly aligned; poly(ethylene glycol) (PEG), a poly(aspartamide) derivative with a 1,2-diaminoethane moiety (PAsp(DET)), and poly(l-lysine). Materials and Methods  The size and in vitro transfection efficacy of the polyplex micelles were determined by dynamic light scattering (DLS) and luciferase assay, respectively. The systemic gene delivery with the polyplex micelles was evaluated from enhanced green fluorescence protein (EGFP) expression in the tumor tissues. Results  The polyplex micelles were approximately 80 nm in size and had one order of magnitude higher in vitro transfection efficacy than that of a diblock copolymer as a control. With the aid of transforming growth factor (TGF)-β type I receptor (TβR-1) inhibitor, which enhances accumulation of macromolecular drugs in tumor tissues, the polyplex micelle from the triblock copolymer showed significant EGFP expression in the pancreatic tumor (BxPC3) tissues, mainly in the stromal regions including the vascular endothelial cells and fibroblasts. Conclusion  The three-layered polyplex micelles were confirmed to be an effective gene delivery system to subcutaneously implanted pancreatic tumor tissues through systemic administration.     

Poly(ε-caprolactone)-<Emphasis Type="Italic">Block</Emphasis>-poly(ethyl Ethylene Phosphate) Micelles for Brain-Targeting Drug Delivery: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Valuation

Purpose  

The purpose of this work was to investigate the potential of poly(ε-caprolactone)-block-poly(ethyl ethylene phosphate) (PCL-PEEP) micelles for brain-targeting drug delivery.     

Comparison Between Hot-Melt Extrusion and Spray-Drying for Manufacturing Solid Dispersions of the Graft Copolymer of Ethylene Glycol and Vinylalcohol

Purpose  

To investigate the effect of the manufacturing method (spray-drying or hot-melt extrusion) on the kinetic miscibility of miconazole and the graft copolymer poly(ethyleneglycol-g-vinylalcohol). The effect of heat pre-treatment of solutions used for spray-drying and the use of spray-dried copolymer as excipient for hot-melt extrusion was investigated.     

Micelles of Different Morphologies—Advantages of Worm-like Filomicelles of PEO-PCL in Paclitaxel Delivery

Purpose

Worm-like and spherical micelles are both prepared here from the same amphiphilic diblock copolymer, poly(ethylene oxide)-b-poly (ε-caprolactone) (PEO [5 kDa]–PCL [6.5 kDa]) in order to compare loading and delivery of hydrophobic drugs.

Materials and Methods

Worm-like micelles of this degradable copolymer are nanometers in cross-section and spontaneously assemble to stable lengths of microns, resembling filoviruses in some respects and thus suggesting the moniker ‘filomicelles’. The highly flexible worm-like micelles can also be sonicated to generate kinetically stable spherical micelles composed of the same copolymer.

Results

The fission process exploits the finding that the PCL cores are fluid, rather than glassy or crystalline, and core-loading of the hydrophobic anticancer drug delivery, paclitaxel (TAX) shows that the worm-like micelles load and solubilize twice as much drug as spherical micelles. In cytotoxicity tests that compare to the clinically prevalent solubilizer, Cremophor^® EL, both micellar carriers are far less toxic, and both types of TAX-loaded micelles also show fivefold greater anticancer activity on A549 human lung cancer cells.

Conclusion

PEO–PCL based worm-like filomicelles appear to be promising pharmaceutical nanocarriers with improved solubilization efficiency and comparable stability to spherical micelles, as well as better safety and efficacy in vitro compared to the prevalent Cremophor^® EL TAX formulation.

     

Prolonged Local Anesthetic Action Through Slow Release from Poly(Lactic Acid Co Castor Oil)

Purpose  To evaluate a new formulation of bupivacaine loaded in an injectable fatty acid based biodegradable polymer poly(lactic acid co castor oil) in prolonging motor and sensory block when injected locally. Materials and methods  The polyesters were synthesized from dl-lactic acid and castor oil with feed ratio of 4:6 and 3:7 w/w. Bupivacaine was dispersed in poly(fatty ester) liquid and tested for drug release in vitro. The polymer p(DLLA:CO) 3:7 loaded with 10% bupivacaine was injected through a 22G needle close to the sciatic nerve of ICR mice and the duration of sensory and motor nerve blockade was measured. Results  The dl-lactic acid co castor oil p(DLLA:CO) 3:7 released 65% of the incorporated bupivacaine during 1 week in vitro. Single injection of 10% bupivacaine loaded into this polymer caused motor block that lasted 24 h and sensory block that lasted 48 h. Conclusion  Previously we developed a ricinoleic acid based polymer with incorporated bupivacaine which prolonged anesthesia to 30 h. The new polymer poly(lactic acid co castor oil) 3:7 provides slow release of effective doses of the incorporated local anesthetic agent and prolongs anesthesia to 48 h.     

Novel Polymeric Micelles Based on the Amphiphilic Diblock Copolymer Poly(N-vinyl-2-pyrrolidone)-block-poly(D,L-lactide)

Purpose. The purpose of this work was to synthesize a new amphiphilic diblock copolymer of poly(N-vinyl-2-pyrrolidone and poly(D,L-lactide) (PVP-b-PDLLA) capable of self-assembling into polymeric micelles with multiple binding sites and high entrapment efficiency. Methods. The copolymer was synthesized by ring-opening polymerization of D,L-lactide initiated by potassium PVP hydroxylate. It was characterized by gel permeation chromatography, ¹H- and ¹³C-NMR spectroscopy. The ability of the copolymer to self-assemble was demonstrated by dynamic and static light scattering, spectrofluorimetry and ¹H-NMR. The hydrophobic model drug indomethacin was incorporated into the polymeric micelles by a dialysis procedure. Results. A series of amphiphilic diblock copolymers based on PVP-b-PDLLA were successfully synthesized. The critical association concentrations in water were low, always below 15 mg/L. Micellar size was generally bimodal with a predominant population between 40 and 100 nm. PVP-b-PDLLA micelles were successfully loaded with the poorly water-soluble drug indomethacin and demonstrated an entrapment efficiency higher than that observed with control poly(ethylene glycol)-b-PDLLA micelles. It was hypothesized that specific interactions with the hydrophilic outer shell could contribute to the increase in drug loading. Conclusion. PVP-b-PDLLA micelles appear to exhibit multiple binding sites and thus represent a promising strategy for the delivery of a variety of drugs.     

Evaluation of Temperature-Sensitive,Indocyanine Green-Encapsulating Micelles for Noninvasive Near-Infrared Tumor Imaging

Purpose  

Indocyanine green (ICG), an FDA-approved near infrared (NIR) dye, has potential application as a contrast agent for tumor detection. Because ICG binds strongly to plasma proteins and exhibits aqueous, photo, and thermal instability, its current applications are largely limited to monitoring blood flow. To address these issues, ICG was encapsulated and stabilized within polymeric micelles formed from the thermo-sensitive block copolymer Pluronic F-127, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), to increase the stability and circulation time of ICG.     

In vitro controlled release of clove essential oil in self-assembly of amphiphilic polyethylene glycol-block-polycaprolactone

In this study, a micellar delivery system with an amphiphilic diblock copolymer of poly (ethylene glycol) and poly (?-caprolactone) was synthesised and used to incorporate hydrophobic clove essential oil (CEO). To determine an optimal delivery system, the effects of the copolymer’s hydrophobic block length and the CEO-loading content on the encapsulation of CEO were investigated. Percentages of entrapment efficiency (%EE), CEO loading (%CEO), and in vitro release profiles were determined. The size, size distribution, zeta potential, and morphology of the obtained micelles were determined by DLS, FE-SEM, and TEM. The %EE, %CEO, and in vitro release profiles of CEO incorporated in micelles were analysed by HPLC. The study revealed a sustained release profile of CEO from CEO-loaded micelles. The results indicate the successful formulation of CEO-loaded PEG-b-PCL micelle nanoparticles. It is suggested that this micelle system has considerably potential applications in the sustained release of CEO in intravascular drug delivery.     

Synthesis, antiplatelet aggregation activity, and molecular modeling study of novel substituted-piperazine analogues

Abstract  

New carbamoylpyridine and carbamoylpiperidine analogues containing nipecotic acid scaffold were designed, synthesized, and evaluated for their platelet aggregation inhibitory activity. Molecular modeling investigation was performed and the impact of lipophilicity on activity was also discussed. Structure activity relationship among this series was obtained. N ¹-[1-(4-bromobenzyl)-3-piperidino-carbonyl]-N ⁴-(2-chlorophenyl)-piperazine hydrobromide (20), and 1,4-bis-[3-[N ⁴-(2-chlorophenyl)-N ¹-(piperazino-carbonyl)]-piperidin-1-yl-methyl]-benzene dibromide (30) are the most active antiplatelet aggregating compounds in this study, both at concentration of 0.06 μM.     

Topical delivery of retinol emulsions co-stabilised by PEO-PCL-PEO triblock copolymers: effect of PCL block length

This article describes enhanced skin permeation and UV/thermal stability of retinol emulsions by the co-stabilisation of Tween20 and biodegradable poly(ethylene oxide)-block-poly(ε-caprolactone)-block-poly(ethylene oxide) (PEO-PCL-PEO) triblock copolymers having different lengths of hydrophobic PCL block. A triblock copolymer with a longer PCL block has a lower hydrophile-lipophile balance (HLB) value. Commercial Retinol 50C® (BASF Co., Ludwigshafen, Germany) was used as the source of retinol. Ultrasonication of the Retinol 50C® emulsion with the triblock copolymers led to an increase in retinol solubilisation and a decrease in average particle size of the resulting retinol emulsion. These characteristics improved skin permeation of retinol through the stratum corneum of artificial skin and subsequent proliferation of viable epidermis cell. Employment of the triblock copolymer with a longer PCL block increased both UV and thermal stabilization of the retinol. These results suggest that HLB and PCL block length are important factors to enhance the topical delivery of retinol into the skin.     

All-trans-retinoic acid release from core-shell type nanoparticles of poly(epsilon-caprolactone)/poly(ethylene glycol) diblock copolymer

Poly(epsilon-caprolactone)/poly(ethylene glycol) (abbreviated as CE) diblock copolymers were synthesized to make core-shell type nanoparticles for all-trans-retinoic acid (atRA). Fluorescence spectroscopy showed that critical association concentration (CAC) value decreased at higher MW of CE diblock copolymer. Drug loading characteristics were studied under various experimental conditions. Drug contents and loading efficiency increased as the MW of poly(epsilon-caprolactone) (PCL) block of CE and initial drug feeding amount increased. Solvent used and preparation method also affected drug contents and loading efficiency. According to 1H NMR using CDCl3 and D2O, specific peaks of the PCL block and drug appearing in CDCl3, disappeared at D2O, suggesting hydrophobic core with hydrophilic shell formed in water. atRA release was faster at smaller MW of copolymer and lower drug contents. Nanoparticles prepared in DMF showed faster release rate compared with those prepared in THF or acetone. Cytotoxicity of atRA against U87MG, U251MG and U343MG cell lines were increased by nanoencapsulation while empty nanoparticles of CE diblock copolymer were not significantly affected.     

Block Copolymer Micelles for Controlled Delivery of Glycolytic Enzyme Inhibitors

Purpose  

To develop block copolymer micelles as an aqueous dosage form for a potent glycolytic enzyme inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO).     

Control of enteric neuromuscular functions by purinergic A3 receptors in normal rat distal colon and experimental bowel inflammation

Background and purpose:

Elephantopus scaber L. (Asteraceae) is a traditional herbal medicine with anti-cancer effects. We evaluated the in vitro and in vivo efficacy of a major sesquiterpene lactone constituent of E. scaber, deoxyelephantopin (DET), against mammary adenocarcinoma and the underlying molecular mechanism.

Experimental approach:

A variety of cellular assays, immunoblotting and immunohistochemistry, as well as both orthotopic and metastatic TS/A tumour models in BALB/c mice, were used. Test mice were pretreated and post-treated with DET or paclitaxel and mammary tumour growth evaluated.

Key results:

DET (≤2 µg·mL⁻¹) significantly inhibited colony formation, cell proliferation, migration and invasion of TS/A cells and induced G₂/M arrest and apoptosis in TS/A cells. c-Jun N-terminal kinase-mediated p21^Waf1/Cip1 expression and caspase activation cascades were up-regulated by DET, effects suppressed by N-acetyl-L-cysteine. Moreover, tumour necrosis factor α-induced matrix metalloproteinase-9 enzyme activity and expression and nuclear factor-kappa B activation were abolished by DET. Pretreatment with DET was more effective than paclitaxel, for profound suppression of orthotopic tumour growth (99% vs. 68% reduction in tumour size) and lung metastasis of TS/A cells (82% vs. 63% reduction in metastatic pulmonary foci) and prolonged median survival time (56 vs. 37 days, P < 0.01) in mice. The levels of cyclooxygenase-2 and vascular endothelial growth factor in metastatic lung tissues of TS/A-bearing mice were attenuated by DET.

Conclusions and implications:

Our data provide evidence for the suppression of mammary adenocarcinoma by DET with several mechanisms and suggest that DET has potential as a chemopreventive agent for breast cancer.     

Biodegradable hybrid recombinant block copolymers for non-viral gene transfection

Thermal targeting of therapeutic genes can enhance local gene concentration to maximize their efficacy. However, lack of safe and efficient carriers has impeded the development of this delivery option. Herein, we report the preparation and evaluation of a hybrid recombinant material, p[Asp(DET)](53)ELP(1-90), that possess a thermo-responsive elastin-like polypeptide (ELP) segment and a diethylenetriamine (DET) modified poly-L-aspartic acid segment. The term, hybrid, indicates that the material was prepared by genetic engineering and synthetic chemistry. In summary, the thermal phase transition behavior and cytotoxicity of the biodegradable copolymer were studied. The polyplexes formed by the copolymer and pGL4 plasmid were characterized by dynamic light scattering and ζ-potential measurements. The polyplexes retained the thermal phase transition behavior conferred by the copolymer; however, they exhibited a two-step transition process not seen with the copolymer. The polyplexes also showed appreciable transfection efficiency with low cytotoxicity.     

Porphyrin and Galactosyl Conjugated Micelles for Targeting Photodynamic Therapy

Purpose  

To study the targeting and photodynamic therapy efficiency of porphyrin and galactosyl conjugated micelles based on amphiphilic copolymer galactosyl and mono-aminoporphyrin (APP) incoporated poly(2-aminoethyl methacrylate)-polycaprolactone (Gal-APP-PAEMA-PCL).     

Stealth Amphotericin B nanoparticles for oral drug delivery: In vitro optimization

Purpose

Amphotericin B (AmB) is an effective anti-fungal and anti-leishmanial agent. However, AmB has low oral bioavailability (0.3%) and adverse effects (e.g., nephrotoxicity). The objectives of this study were to improve the oral bioavailability by entrapping AmB in pegylated (PEG) poly lactide co glycolide copolymer (PLGA–PEG) nanoparticles (NPs). The feasibility of different surfactants and stabilizers on the mean particle size (MPS) and entrapment efficiency were also investigated.

Materials and methods

NPs of AmB were prepared by a modified emulsification diffusion method employing a vitamin E derivative as a stabilizer. Physicochemical properties and particle size characterization were evaluated using Fourier Transform Infra-Red spectroscopy (FTIR), differential scanning calorimetry, scanning electron microscopy and transmission electron microscopy. Moreover, in vitro dissolution profiles were performed for all formulated AmB NPs.

Results

MPS of the prepared spherical particles of AmB ranged from 26.4 ± 2.9 to 1068 ± 489.8 nm. An increased stirring rate favored AmB NPs with a smaller MPS. There was a significant reduction in MPS, drug content and drug release, when AmB NPs were prepared using the diblock polymer PLGA–PEG with 15% PEG. Addition of three emulsifying agents poly vinyl pyrrolidone (PVP), Vitamin E (TPGS) and pluronic F-68 to AmB formulations led to a significant reduction in particle size and increase in drug entrapment efficiency (DEE) compared to addition of PVP alone. FTIR spectroscopy demonstrated a successful loading of AmB to pegylated PLGA–PEG copolymers. PLGA–PEG copolymer entrapment efficiency of AmB was increased up to 56.7%, with 92.7% drug yield. After a slow initial release, between 20% and 54% of AmB was released in vitro within 24 h phosphate buffer containing 2% sodium deoxycholate and were best fit Korsmeyer–Peppas model. In conclusion, PLGA–PEG diblock copolymer with 15% PEG produced a significant reduction (>70%) in MPS with highest drug content. The percentage of PEG in the copolymer and the surfactant/stabilizer used had a direct effect on AmB release in vitro, entrapment efficiency and MPS. These developed formulations are feasible, effective and improved alternatives to other carriers for oral delivery of AmB.     

Mesalazine pharmacokinetics and NAT2 phenotype

Background  Mesalazine undergoes extensive metabolism by N-acetylation. While there is some evidence for an involvement of N-acetyltransferase (NAT) type 1, a potential role of NAT type 2 (NAT2) in vivo has not been tested. Methods  In two studies in healthy young Caucasians, NAT2 phenotyping was carried out using a caffeine metabolic ratio in urine 4-6 h postdose. In study A, 1,000 mg mesalazine doses were given thrice daily for 5 days, and urine and blood samples were drawn during the last dosing interval. In study B, a 1,000 mg single dose was given, and samples were taken for 48 h postdose. Pharmacokinetics of mesalazine and N-acetylmesalazine (LC-MS/MS) were calculated by noncompartmental methods. Results  NAT2 phenotype could be allocated unequivocally in 21 slow and 5 rapid acetylators in study A, and in 9 slow and 8 rapid acetylators in study B. Geometric mean (CV%) values in study A for slow [rapid] acetylators were as follows: mesalazine AUC 11.1 μg/mL·h (51%) [12.0 μg/mL·h (52%)], N-acetylmesalazine AUC 27.7 μg/mL·h (32%) [30.5 μg/mL·h (27%)], mesalazine Ae 8.53% (89%) [9.03% (52%)], N-acetylmesalazine Ae 31.4% (46%) [32.2 (41%)]. Values in study B were as follows: mesalazine AUC 3.45 μg/mL·h (113%) [2.36 μg/mL·h (87%)], N-acetylmesalazine AUC 21.3 μg/mL·h (29%) [18.0 μg/mL·h (39%)], mesalazine Ae 0.2% (256%) [0.1% (359%)], N-acetylmesalazine Ae 30.9% (44%) [18.1% (84%)]. Higher AUC and Ae values for mesalazine in steady state study indicate saturation of mesalazine metabolism. Statistics provided no evidence for a true difference in mesalazine pharmacokinetics between slow and rapid acetylators, and no significant correlation between NAT2 activity and any mesalazine pharmacokinetic parameter was found. Conclusion  NAT2 has no major role in human metabolism of mesalazine in vivo.     

Analysis of Micelle Formation of an Adriamycin-Conjugated Poly(Ethylene Glycol)–Poly(Aspartic Acid) Block Copolymer by Gel Permeation Chromatography

The micelle-forming behavior of a drug–block copolymer conjugate adriamycm-conjugated poly(ethylene glycol)–poly(aspartic acid) block copolymer; PEG-P[Asp(ADR)] was analyzed by gel permeation chromatography (GPC). Four compositions of the conjugates were observed to form micellar structures in aqueous media, and their micelle-forming behavior was found to be dependent on the composition and media. These micelles did not reach equilibrium within short time periods like low molecular weight surfactants. One composition formed stable micelles in the presence of serum.     

Comparative Investigations on In Vitro Serum Stability of Polymeric Micelle Formulations

Purpose  

Stability of polymeric micelles upon injection is essential for a drug delivery system but is not fully understood. We optimized an analytical test allowing quantification of micellar stability in biofluids and applied it to a variety of block copolymer micelles with different hydrophobic block architechtures.