Development of an Acid-Labile Ketal Linked Amphiphilic Block Copolymer Nanoparticles for pH-Triggered Release of Paclitaxel.

Here, we report on the construction of biodegradable poly(ethylene oxide monomethyl ether) (MPEO)-b-poly(ε-caprolactone) (PCL) nanoparticles (NPs) having acid-labile (acyclic ketal group) linkage at the block junction. In the presence of acidic pH, the nanoassemblies were destabilized as a consequence of cleaving this linkage. The amphiphilic MPEO-b-PCL diblock copolymer self-assembled in PBS solution into regular spherical NPs. The structure of self-assemble and disassemble NPs were characterized in detail by dynamic (DLS), static (SLS) light scattering, small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). The key of the obtained NPs is using them in a paclitaxel (PTX) delivery system and study their in vitro cytostatic activity in a cancer cell model. The acid-labile ketal linker enabled the disassembly of the NPs in a buffer simulating an acidic environment in endosomal (pH ~5.0 to ~6.0) and lysosomal (pH ~4.0 to ~5.0) cell compartments resulting in the release of paclitaxel (PTX) and formation of neutral degradation products. The in vitro cytotoxicity studies showed that the activity of the drug-loaded NPs was increased compared to the free PTX. The ability of the NPs to release the drug at the endosomal pH with concomitant high cytotoxicity makes them suitable candidates as a drug delivery system for cancer therapy.

Paclitaxel-loaded biodegradable ROS-sensitive nanoparticles for cancer therapy.

BACKGROUND: Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, trigger biodegradation of polymer-based nanoparticles (NPs) bearing pinacol-type boronic ester groups. These NPs may selectively release their cargo, in this case paclitaxel (PTX), at the high levels of ROS present in the intracellular environment of inflamed tissues and most tumors. PURPOSE: The main objective was to determine anti-tumor efficacy of PTX-loaded ROS-sensitive NPs and to examine whether macrophage infiltration had any impact on treatment efficacy. METHODS: NPs were synthesized and their characteristics in the presence of H2O2 were demonstrated. Both confocal microscopy as well as flow cytometry approaches were used to determine degradation of ROS-sensitive NPs. HeLa cells were cultured in vitro and used to establish tumor xenografts in nude mice. In vivo experiments were performed to understand toxicity, biodistribution and anti-tumor efficacy of the NPs. Moreover, we performed immunohistochemistry on tumor sections to study infiltration of M1 and M2 subsets of macrophages. RESULTS: We demonstrated that PTX delivered in NPs containing a ROS-sensitive polymer exhibits a better anti-tumor efficacy than PTX in NPs containing ROS-non-sensitive polymer, free PTX or Abraxane® (nab-PTX). The biodistribution revealed that ROS-sensitive NPs exhibit retention in liver, spleen and lungs, suggesting a potential to target cancer metastasizing to these organs. Finally, we demonstrated a correlation between infiltrated macrophage subsets and treatment efficacy, possibly contributing to the efficient anti-tumor effects. CONCLUSION: Treatment with ROS-sensitive NPs containing PTX gave an improved therapeutic effect in HeLa xenografts than their counterpart, free PTX or nab-PTX. Our data revealed a correlation between macrophage infiltration and efficiency of the different antitumor treatments, as the most effective NPs resulted in the highest infiltration of the anti-tumorigenic M1 macrophages.

Zwitterionic Functionalizable Scaffolds with Gyroid Pore Architecture for Tissue Engineering.

Stereolithography-assisted fabrication of hydrogels of carboxybetaine methacrylamide (CBMAA) and a α,ω-methacrylate poly(d,l-lactide-block-ethylene glycol-block- d,l-lactide) (MA-PDLLA-PEG-PDLLA-MA) telechelic triblock macromer is presented. This technique allows printing complex structures with gyroid interconnected porosity possessing extremely high specific area. Hydrogels are characterized by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and laser scanning confocal microscopy (LSCM). The copolymerization with zwitterionic comonomer leads hydrogels with high equilibrium water content (EWC), up to 700% while maintaining mechanical robustness. The introduction of carboxybetaine yields excellent resistance to nonspecific protein adsorption while providing a facile way for specific biofunctionalization with a model protein, fluorescein isothiocyanate labeled bovine serum albumin (BSA). The homogeneous protein immobilization across the hydrogel pores prove the accessibility to the innermost pore volumes. The remarkably low protein adsorption combined with the interconnected nature of the porosity allowing fast diffusion of nutrient and waste product and the mimicry of bone trabecular, makes the hydrogels presented here highly attractive for tissue engineering.

Poly(ethylene oxide monomethyl ether)- block-poly(propylene succinate) Nanoparticles: Synthesis and Characterization, Enzymatic and Cellular Degradation, Micellar Solubilization of Paclitaxel, and in Vitro and in Vivo Evaluation.

Polyester-based nanostructures are widely studied as drug-delivery systems due to their biocompatibility and biodegradability. They are already used in the clinic. In this work, we describe a new and simple biodegradable and biocompatible system as the Food and Drug Administration approved polyesters (poly-ε-caprolactone, polylactic acid, and poly(lactic- co-glycolic acid)) for the delivery of the anticancer drug paclitaxel (PTX) as a model drug. A hydrophobic polyester, poly(propylene succinate) (PPS), was prepared from a nontoxic alcohol (propylene glycol) and monomer from the Krebs's cycle (succinic acid) in two steps via esterification and melt polycondensation. Furthermore, their amphiphilic block copolyester, poly(ethylene oxide monomethyl ether)- block-poly(propylene succinate) (mPEO- b-PPS), was prepared by three steps via esterification followed by melt polycondensation and the addition of mPEO to the PPS macromolecules. Analysis of the in vitro cellular behavior of the prepared nanoparticle carriers (NPs) (enzymatic degradation, uptake, localization, and fluorescence resonance energy-transfer pair degradation studies) was performed by fluorescence studies. PTX was loaded to the NPs of variable sizes (30, 70, and 150 nm), and their in vitro release was evaluated in different cell models and compared with commercial PTX formulations. The mPEO- b-PPS copolymer analysis displays glass transition temperature < body temperature < melting temperature, lower toxicity (including the toxicity of their degradation products), drug solubilization efficacy, stability against spontaneous hydrolysis during transport in bloodstream, and simultaneous enzymatic degradability after uptake into the cells. The detailed cytotoxicity in vitro and in vivo tumor efficacy studies have shown the superior efficacy of the NPs compared with PTX and PTX commercial formulations.

Thermoresponsive ß-glucan-based polymers for bimodal immunoradiotherapy - Are they able to promote the immune system?

A conceptually new bimodal immunoradiotherapy treatment was demonstrated using thermoresponsive polymer ß-glucan-graft-poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline) bearing complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with yttrium-90(III) at the graft ends. The behavior of this thermoresponsive polymer in aqueous solutions was studied, and it showed the appropriate cloud point temperature for brachytherapy applications. The polymer was tested in vitro, and it exhibited nontoxicity and active uptake into cancer cells and macrophages with colocalization in the lysosomes and macrophagosomes. Moreover, the observed oxidative burst response of the leukocytes established the immunostimulatory properties of the polymer, which were also studied in vivo after injection into the thigh muscles of healthy mice. The subsequent histological evaluation revealed the extensive immune activation reactions at the site of injection. Furthermore, the production of tumor necrosis factor α induced by the prepared polymer was observed in vitro, denoting the optimistic prognosis of the treatment. The biodistribution study in vivo indicated the formation of the polymer depot, which was gradually degraded and excluded from the body. The radiolabeled polymer was used during in vivo antitumor efficiency experiments on mice with EL4 lymphoma. The immunoradiotherapy group (treated with the radiolabeled polymer) demonstrated the complete inhibition of tumor growth during the beginning of the treatment. Moreover, 7 of the 15 mice were completely cured in this group, while the others exhibited significantly prolonged survival time compared to the control group. The in vivo experiments indicated the considerable synergistic effect of using immunoradiotherapy compared to separately using immunotherapy or radiotherapy.

Poly(2-ethyl-2-oxazoline) conjugates with doxorubicin for cancer therapy: In vitro and in vivo evaluation and direct comparison to poly[N-(2-hydroxypropyl)methacrylamide] analogues.

We designed and synthesized a new delivery system for the anticancer drug doxorubicin based on a biocompatible hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx) carrier with linear architecture and narrow molar mass distribution. The drug is connected to the polymer backbone via an acid-sensitive hydrazone linker, which allows its triggered release in the tumor. The in vitro studies demonstrate successful cellular uptake of conjugates followed by release of the cytostatic cargo. In vivo experiments in EL4 lymphoma bearing mice revealed prolonged blood circulation, increased tumor accumulation and enhanced antitumor efficacy of the PEtOx conjugate having higher molecular weight (40 kDa) compared to the lower molecular weight (20 kDa) polymer. Finally, the in vitro and in vivo anti-cancer properties of the prepared PEtOx conjugates were critically compared with those of the analogous system based on the well-established PHPMA carrier. Despite the relatively slower intracellular uptake of PEtOx conjugates, resulting also in their lower cytotoxicity, there are no substantial differences in in vivo biodistribution and anti-cancer efficacy of both classes of polymer-Dox conjugates. Considering the synthetic advantages of poly(2-alkyl-2-oxazoline)s, the presented study demonstrates their potential as a versatile alternative to well-known PEO- or PHPMA-based materials for construction of drug delivery systems.

Fluorescent boronate-based polymer nanoparticles with reactive oxygen species (ROS)-triggered cargo release for drug-delivery applications.

A new drug-delivery system of polymer nanoparticles (NPs) bearing pinacol-type boronic ester and alkyne moieties displaying triggered self-immolative polymer degradation in the presence of reactive oxygen species (ROS) with the capability of cellular imaging is presented. The NPs specifically release their drug cargo under concentrations of ROS that are commonly found in the intracellular environment of certain tumors and of inflamed tissues and exhibit significant cytotoxicity to cancer cells compared to their non-ROS-responsive counterparts.

Absolute quantitation of sub-micrometer particles in cells by flow cytometry.

Absolute quantitative measurements of nanoparticle (NP) uptake are a prerequisite to determine doses of NPs in pharmacological and toxicological studies. However, absolute quantitation is rarely reported, hindering the comparison between different studies. Here, a new flow cytometric approach is presented to analyze fluorescent NPs with a "standard" non-scanning flow cytometer and to quantify them inside cells. The mean fluorescence intensity of a single particle and the particle concentration (NPs per µL medium) are obtained. A routine for rapid quantitative counting of the endocytosed NPs in HeLa cells by flow cytometry (FC) is developed and validated by confocal laser scanning microscopy. As a proof-of-concept, the quantitative measurements show that the cellular uptake efficiency of negatively charged poly(methyl methacrylate) NPs is very low, that is, in the range of 10(-3) % of the added particle amount.

Competitive cellular uptake of nanoparticles made from polystyrene, poly(methyl methacrylate), and polylactide.

The uptake behavior of negatively charged fluorescent nanoparticles made from different polymers (PS, PMMA, and PLLA) is studied on HeLa cells. All particles are obtained by the miniemulsion process using sodium dodecylsulfate as anionic surfactant. The size of the particles is in the range 105-125 nm. Cell uptake is analyzed by flow cytometry and reveals a higher uptake of PLLA particles compared to PMMA and PS particles. In competitive uptake studies two different types of particles are co-incubated with the HeLa cells; the results indicate a mutual influence of the particles on their uptake behavior. A reduced internalization of PLLA particles in the presence of PS particles is observed, although neither the co-incubation of PMMA and PLLA nor of PMMA and PS shows similar effect.

Synthesis of fluorescent polyisoprene nanoparticles and their uptake into various cells.

Fluorescent polyisoprene nanoparticles were synthesized by the miniemulsion technique as marker particles for cells. The uptake of the non-functionalized polyisoprene nanoparticles, without any transfection agents, into different adherent (HeLa) and also suspension (Jurkat) cell lines is strikingly efficient and fast compared to other polymeric particles, and leads to high loading of the cells. The intracellular polyisoprene particles are localized as single particles in endosomes distributed throughout the entire cytoplasm. The uptake kinetics shows that particle internalization starts during the first minutes of incubation and is finished after 48 h of incubation. Since (unfunctionalized) polystyrene particles show a comparable, low uptake behavior in cells, the uptake rates can be tuned by the amount of polystyrene in polyisoprene/polystyrene copolymer particles. As polyisoprene nanoparticles are internalized by different cell lines that are relevant for biomedical applications, they can be used to label these cells efficiently if a marker is incorporated in the particles. As polyisoprene is not or is hardly biodegradable the particles should be suited for long-term applications.