Membrane Permeability and Responsiveness Drive Performance: Linking Structural Features with the Antitumor Effectiveness of Doxorubicin-Loaded Stimuli-Triggered Polymersomes.

The permeability and responsiveness of polymer membranes are absolutely relevant in the design of polymersomes for cargo delivery. Accordingly, we herein correlate the structural features, permeability, and responsiveness of doxorubicin-loaded (DOX-loaded) nonresponsive and stimuli-responsive polymersomes with their in vitro and in vivo antitumor performance. Polymer vesicles were produced using amphiphilic block copolymers containing a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) segment linked to poly[N-(4-isopropylphenylacetamide)ethyl methacrylate] (PPPhA, nonresponsive block), poly[4-(4,4,5,5-tetra-methyl-1,3,2-dioxaborolan-2-yl)benzyl methacrylate] [PbAPE, reactive oxygen species (ROS)-responsive block], or poly[2-(diisopropylamino)ethyl methacrylate] (PDPA, pH-responsive block). The PDPA-based polymersomes demonstrated outstanding biological performance with antitumor activity notably enhanced compared to their counterparts. We attribute this behavior to a fast-triggered DOX release in acidic tumor environments as induced by pH-responsive polymersome disassembly at pH < 6.8. Possibly, an insufficient ROS concentration in the selected tumor model attenuates the rate of ROS-responsive vesicle degradation, whereas the nonresponsive nature of the PPPhA block remarkably impacts the performance of such potential nanomedicines.

Reactive Oxygen Species (ROS)-Responsive Polymersomes with Site-Specific Chemotherapeutic Delivery into Tumors via Spacer Design Chemistry.

The lack of cellular and tissue specificities in conventional chemotherapies along with the generation of a complex tumor microenvironment (TME) limits the dosage of active agents that reaches tumor sites, thereby resulting in ineffective responses and side effects. Therefore, the development of selective TME-responsive nanomedicines is of due relevance toward successful chemotherapies, albeit challenging. In this framework, we have synthesized novel, ready-to-use ROS-responsive amphiphilic block copolymers (BCs) with two different spacer chemistry designs to connect a hydrophobic boronic ester-based ROS sensor to the polymer backbone. Hydrodynamic flow focusing nanoprecipitation microfluidics (MF) was used in the preparation of well-defined ROS-responsive PSs; these were further characterized by a combination of techniques [1H NMR, dynamic light scattering (DLS), static light scattering (SLS), transmission electron microscopy (TEM), and cryogenic TEM (cryo-TEM)]. The reaction with hydrogen peroxide releases an amphiphilic phenol or a hydrophilic carboxylic acid, which affects polymersome (PS) stability and cargo release. Therefore, the importance of the spacer chemistry in BC deprotection and PS stability and cargo release is herein highlighted. We have also evaluated the impact of spacer chemistry on the PS-specific release of the chemotherapeutic drug doxorubicin (DOX) into tumors in vitro and in vivo. We demonstrate that by spacer chemistry design one can enhance the efficacy of DOX treatments (decrease in tumor growth and prolonged animal survival) in mice bearing EL4 T cell lymphoma. Side effects (weight loss and cardiotoxicity) were also reduced compared to free DOX administration, highlighting the potential of the well-defined ROS-responsive PSs as TME-selective nanomedicines. The PSs could also find applications in other environments with high ROS levels, such as chronic inflammations, aging, diabetes, cardiovascular diseases, and obesity.

Surface Design of Antifouling Vascular Constructs Bearing Biofunctional Peptides for Tissue Regeneration Applications.

Antifouling polymer layers containing extracellular matrix-derived peptide motifs offer promising new options for biomimetic surface engineering. In this contribution, we report the design of antifouling vascular grafts bearing biofunctional peptide motifs for tissue regeneration applications based on hierarchical polymer brushes. Hierarchical diblock poly(methyl ether oligo(ethylene glycol) methacrylate-block-glycidyl methacrylate) brushes bearing azide groups (poly(MeOEGMA-block-GMA-N3)) were grown by surface-initiated atom transfer radical polymerization (SI-ATRP) and functionalized with biomimetic RGD peptide sequences. Varying the conditions of copper-catalyzed alkyne-azide "click" reaction allowed for the immobilization of RGD peptides in a wide surface concentration range. The synthesized hierarchical polymer brushes bearing peptide motifs were characterized in detail using various surface sensitive physicochemical methods. The hierarchical brushes presenting the RGD sequences provided excellent cell adhesion properties and at the same time remained resistant to fouling from blood plasma. The synthesis of anti-fouling hierarchical brushes bearing 1.2 × 103 nmol/cm2 RGD biomimetic sequences has been adapted for the surface modification of commercially available grafts of woven polyethylene terephthalate (PET) fibers. The fiber mesh was endowed with polymerization initiator groups via aminolysis and acylation reactions optimized for the material. The obtained bioactive antifouling vascular grafts promoted the specific adhesion and growth of endothelial cells, thus providing a potential avenue for endothelialization of artificial conduits.

Impact of Bioactive Peptide Motifs on Molecular Structure, Charging, and Nonfouling Properties of Poly(ethylene oxide) Brushes.

Polymer layers capable of suppressing protein adsorption from biological media while presenting extracellular matrix-derived peptide motifs offer valuable new options for biomimetic surface engineering. Herein, we provide detailed insights into physicochemical changes induced in a nonfouling poly(ethylene oxide) (PEO) brush/polydopamine (PDA) system by incorporation of adhesion ligand (RGD) peptides. Brushes with high surface chain densities (σ ≥ 0.5 chains·nm-2) and pronounced hydrophilicity (water contact angles ≤ 10°) were prepared by end-tethering of heterobifunctional PEOs ( Mn ≈ 20 000 g·mol-1) to PDA-modified surfaces from a reactive melt. Using alkyne distal end group on the PEO chains, azidopentanoic-bearing peptides were coupled through a copper-catalyzed Huisgen azide-alkyne "click" cycloaddition reaction. The surface concentration of RGD was tuned from complete saturation of the PEO surface with peptides (1.7 × 105 fmol·cm-2) to values which may induce distinct differences in cell adhesion (<6.0 × 102 fmol·cm-2). Infrared reflection-absorption and X-ray photoelectron spectroscopies proved the PDA-PEO layers covalent structure and the immobilization of RGD peptides. The complete reconstruction of experimental electrohydrodynamics data utilizing mean-field theory predictions further verified the attained brush structure of the end-tethered PEO chains which provided hydrodynamic screening of the PDA anchor. Increasing the surface concentration of immobilized RGD peptides led to increased interfacial charging. Supported by simulations, this observation was attributed to the ionization of functional groups in the amino acid sequence and to the pH-dependent adsorption of water ions (OH- > H3O+) from the electrolyte. Despite the distinct differences observed in the electrokinetic analysis of the surfaces bearing different amounts of RGD, it was found that the peptide presence on PEO(20 000)-PDA layers does not have a significant effect on the nonfouling properties of the system. Notably, the presented PEO(20 000)-PDA layers bearing RGD peptides in the surface concentration range 5.9 to 1.7 × 105 fmol·cm-2 reduced the protein adsorption from fetal bovine serum to less than 30 ng·cm-2, that is, values comparable to the ones obtained for pristine PEO(20 000)-PDA layers.

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.

Polyelectrolyte pH-Responsive Protein-Containing Nanoparticles: The Physicochemical Supramolecular Approach.

We report on the physicochemical properties and self-assembly behavior of novel efficient pH-sensitive nanocontainers based on the Food and Drug Administration-approved anionic polymer Eudragit L100-55 (poly(methacrylic acid-co-ethyl acrylate) 1:1) and nonionic surfactant Brij98. The features of the interaction between Eudragit L100-55 and Brij98 at different pH values and their optimal ratio for nanoparticle formation were studied using isothermal titration calorimetry. The influence of the polymer-to-surfactant ratio on the size and structure of particles was studied at different pH values using dynamic light scattering and small-angle X-ray scattering methods. It was shown that stable nanoparticles are formed at acidic pH at polymer-to-surfactant molar ratios from 1:43 to 1:139. Trypsin was successfully encapsulated into Eudragit-Brij98 nanoparticles as a model bioactive component. The loading efficiency was determined by labeling trypsin with radioactive iodine-125. Eudragit-Brij98 nanoparticles effectively protected trypsin against pepsin digestion. The results showed that trypsin encapsulated into novel pH-sensitive nanocontainers retained more than 50% of its activity after treatment with pepsin compared with nonencapsulated trypsin. The described concept will contribute both to understanding the principles of and designing next-generation nanocontainers.

Thermoresponsive Polymers for Nuclear Medicine: Which Polymer Is the Best?

Thermoresponsive polymers showing cloud point temperatures (CPT) in aqueous solutions are very promising for the construction of various systems in biomedical field. In many of these applications these polymers get in contact with ionizing radiation, e.g., if they are used as carriers for radiopharmaceuticals or during radiation sterilization. Despite this fact, radiosensitivity of these polymers is largely overlooked to date. In this work, we describe the effect of electron beam ionizing radiation on the physicochemical and phase separation properties of selected thermoresponsive polymers with CPT between room and body temperature. Stability of the polymers to radiation (doses 0-20 kGy) in aqueous solutions increased in the order poly(N-vinylcaprolactam) (PVCL, the least stable) ≪ poly[N-(2,2-difluoroethyl)acrylamide] (DFP) < poly(N-isopropylacrylamide) (PNIPAM) ≪ poly(2-isopropyl-2-oxazoline-co-2-n-butyl-2-oxazoline) (POX). Even low doses of ß radiation (1 kGy), which are highly relevant to the storage of polymer radiotherapeutics and sterilization of biomedical systems, cause significant increase in molecular weight due to cross-linking (except for POX, where this effect is weak). In the case of PVCL irradiated with low doses, the increase in molecular weight induced an increase in the CPT of the polymer. For PNIPAM and DFP, there is strong chain hydrophilization leading to an increase in CPT. From this perspective, POX is the most suitable polymer for the construction of delivery systems that experience exposure to radiation, while PVCL is the least suitable and PNIPAM and DFP are suitable only for low radiation demands.

Toward structured macroporous hydrogel composites: electron beam-initiated polymerization of layered cryogels.

The ability to tailor mechanical properties and architecture is crucial in creating macroporous hydrogel scaffolds for tissue engineering. In the present work, a technique for the modification of the pore size and stiffness of acrylamide-based cryogels is demonstrated via the regulation of an electron beam irradiation dose. The samples were characterized by equilibrium swelling measurements, light and scanning electron microscopy, mercury porosimetry, Brunauer-Emmett-Teller surface area analysis, and stiffness measurements. Their properties were compared to cryogels prepared by a standard redox-initiated radical polymerization. A (125)I radiolabeled azidopentanoyl-GGGRGDSGGGY-NH2 peptide was bound to the surface to determine the concentration of the adhesive sites available for biomimetic modification. The functionality of the prepared substrates was evaluated by in vitro cultivation of adipose-derived stem cells. Moreover, the feasibility of preparing layered cryogels was demonstrated. This may be the key to the future preparation of complex hydrogel-based scaffolds to mimic the extracellular microenvironment in a wide range of applications.

Fluorescent nanodiamonds embedded in biocompatible translucent shells.

High pressure high temperature (HPHT) nanodiamonds (NDs) represent extremely promising materials for construction of fluorescent nanoprobes and nanosensors. However, some properties of bare NDs limit their direct use in these applications: they precipitate in biological solutions, only a limited set of bio-orthogonal conjugation techniques is available and the accessible material is greatly polydisperse in shape. In this work, we encapsulate bright 30-nm fluorescent nanodiamonds (FNDs) in 10-20-nm thick translucent (i.e., not altering FND fluorescence) silica shells, yielding monodisperse near-spherical particles of mean diameter 66 nm. High yield modification of the shells with PEG chains stabilizes the particles in ionic solutions, making them applicable in biological environments. We further modify the opposite ends of PEG chains with fluorescent dyes or vectoring peptide using click chemistry. High conversion of this bio-orthogonal coupling yielded circa 2000 dye or peptide molecules on a single FND. We demonstrate the superior properties of these particles by in vitro interaction with human prostate cancer cells: while bare nanodiamonds strongly aggregate in the buffer and adsorb onto the cell membrane, the shell encapsulated NDs do not adsorb nonspecifically and they penetrate inside the cells.

Silver-coated monolithic columns for separation in radiopharmaceutical applications.

In this study, we demonstrate the preparation of a macroporous monolithic column containing anchored silver nanoparticles and its use for the elimination of excess radioiodine from the radiolabeled pharmaceutical. The poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith was first functionalized with cystamine and the free thiol groups liberated by reaction with borohydride. In-house-prepared silver nanoparticles were then attached by interaction with the surface thiols. The deiodization process was demonstrated with the commonly used radiopharmaceutical m-iodobenzylguanidine labeled with radionuclide iodine-125.

Biodistribution study of 211Pb progeny released from intravenously applied 223Ra labelled TiO2 nanoparticles in a mouse model.

BACKGROUND: Targeted alpha therapy is one of the most powerful therapeutical modalities available in nuclear medicine. It's therapeutic potency is based on the nuclides that emit one or several alpha particles providing strong and highly localized therapeutic effects. However, some of these radionuclides, like e.g.223Ra or 225Ac decay in cascades, where the radioactive progeny originating from the consecutive alpha-decays may leave the original vector and cause unwanted irradiation of non-target organs. This progeny, even if partially retained in target tissues by internalization processes, typically do not follow the fate of originally targeted radiopharmaceutical and potentially spread over body following their own biodistribution. In this study we aimed to estimate 211Pb/211Bi progeny fate from the 223Ra surface-labelled TiO2 nanoparticles in vitro and the fate of 211Pb in vivo in a mice model. RESULTS: In vitro stability studies have shown significant differences between the release of the mother 223Ra and its progeny (211Pb, 211Bi) in all the biological matrices that have been tested. The lowest released activities were measured in saline, resulting in less than 5 % of released activity for all nuclides. Contrary to that, the highest released activity of 223Ra of up to 10 % within 48 h was observed in 5 % solution of albumin. The released activity of its progeny; the 211Pb and 211Bi was in the range of 20-40 % in this test medium. Significantly higher released activities of 211Pb and 211Bi compared to 223Ra by at least 10 % was observed in each biological medium, except saline, where no significant differences were observed. The in vivo biodistribution studies results in a mice model, show similar pattern, where it was found that even after accumulation of nanoparticles in target tissues, approximately 10 % of 211Pb is continuously released into the blood stream within 24 h, followed by its natural accumulation in kidneys. CONCLUSION: This study confirms our assumption that the progeny formed in a chain alpha decay of a certain nuclide, in this case the 223Ra, can be released from its original vector, leave the target tissue, relocate and could be deposited in non-target organs. We did not observe complete progeny wash-out from its original target tissues in our model. This indicates strong dependence of the progeny hot atom fate after its release from the original radiopharmaceutical preparation on multiple factors, like their internalization and retention in cells, cell membranes, extracellular matrices, protein binding, etc. We hypothesize, that also the primary tumour or metastasis size, their metabolic activity may significantly influence progeny fate in vivo, directly impacting the dose delivered to non-target tissues and organs. Therefore a bottom-up approach should be followed and detailed pre-/clinical studies on the release and biodistribution of radioactive progeny originating from the chain alpha emitters should be preferably performed.

Phototriggered Cytotoxicity of Ferrocene-Based Micelles - A Nonconventional Approach to Phototherapy.

We report the design, synthesis, and in vitro evaluation of stimuli-responsive nanoscale micelles that can be activated by light to induce a cytotoxic effect. Micelles were assembled from amphiphilic units made of a photoactivatable ferrocenyl linker, connected on one side to a lipophilic chain, and on the other side to a hydrophilic pegylated chain. In vitro experiments indicated that pristine micelles ("off" state) were nontoxic to MCF-7 cancer cells, even at high concentrations, but became potent upon photoactivation ("on" state). The illumination process led to the dissociation of the micelles and the concomitant release of iron species, triggering cytotoxicity.

Tuning polymer-blood and polymer-cytoplasm membrane interactions by manipulating the architecture of poly(2-oxazoline) triblock copolymers.

Bioactive moieties designed to bind to cell membrane receptors benefit from coupling with polymeric carriers that have enhanced affinity to the cell membrane. When bound to the cell surface, such carriers create a "2D solution" of a ligand with a significantly increased concentration near a membrane-bound receptor compared to a freely water-soluble ligand. Bifunctional polymeric carriers based on amphiphilic triblock copolymers were synthesized from 2-pent-4-ynyl oxazoline, 2-nonyl oxazoline and 2-ethyl oxazoline. Their self-assembly and interactions with plasma proteins and HEK 293 cells were studied in detail. The affinity of these triblock copolymers to HEK 293 cell membranes and organ tissues was tunable by the overall hydrophobicity of the polymer molecule, which is determined by the length of the hydrophobic and hydrophilic blocks. The circulation time and biodistribution of three representative triblock copolymers were monitored after intravenous administration to C57BL/6 albino mice. A prolonged circulation time was observed for polymers with longer hydrophobic blocks, despite their molecular weight being below the renal threshold.

Thermoresponsive nanoparticles based on poly(2-alkyl-2-oxazolines) and Pluronic F127.

We synthesized statistical poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazolines) (POXs) that are molecularly dissolved below their cloud point temperature in aqueous milieu and are incorporated into micellar nanoparticles of biocompatible Pluronic F127 (F127) after heating their solution above transition temperature, T(tr). A functional comonomer 2-(but-3-enyl)-2-oxazoline copolymerized into one of the POXs (polymer E) allows introduction of fenolic moieties and subsequent radionuclide labeling with iodine-125. Self-assembly of the polymer E with F127 leads to formation of radioactive nanoparticles with hydrodynamic diameter 20 nm in aqueous solution by heating to 37 °C. The nanoparticles are intended to be used as radioimaging tool in solid tumor diagnostics.

Stimuli-responsive polypeptide nanogels for trypsin inhibition.

A new type of hydrophilic, biocompatible, and biodegradable polypeptide nanogel depots loaded with the natural serine protease inhibitor α1-antitrypsin (AAT) was applied for the inhibition of the inflammatory mediator trypsin. Two types of nanogels were prepared from linear synthetic polypeptides based on biocompatible and biodegradable poly[N 5-(2-hydroxyethyl)-ʟ-glutamine-ran-N 5-propargyl-ʟ-glutamine-ran-N 5-(6-aminohexyl)-ʟ-glutamine]-ran-N 5-[2-(4-hydroxyphenyl)ethyl)-ʟ-glutamine] (PHEG-Tyr) or biocompatible N α-ʟ-lysine-grafted α,ß-poly[(2-propyne)-ᴅ,ʟ-aspartamide-ran-(2-hydroxyethyl)-ᴅʟ-aspartamide-ran-(2-(4-hydroxyphenyl)ethyl)-ᴅʟ-aspartamide] (N α-Lys-NG). Both nanogels were prepared by HRP/H2O2-mediated crosslinking in inverse miniemulsions with pH and temperature-stimuli responsive behavior confirmed by dynamic light scattering and zeta potential measurements. The loading capacity of PHEG-Tyr and N α-Lys-NG nanogels and their release profiles were first optimized with bovine serum albumin. The nanogels were then used for loading and release of AAT. PHEG-Tyr and N α-Lys-NG nanogels showed different loading capacities for AAT with the maximum (20%) achieved with N α-Lys-NG nanogel. In both cases, the nanogel depots demonstrated a burst release of AAT during the first 6 h, which could be favorable for quick inhibition of trypsin. A consequent pilot in vitro inhibition study revealed that both PHEG-Tyr and N α-Lys-NG nanogels loaded with AAT successfully inhibited the enzymatic activity of trypsin. Furthermore, the inhibitory efficiency of the AAT-loaded nanogels was higher than that of only AAT. Interestingly, also non-loaded PHEG-Tyr and N α-Lys-NG nanogels were shown to effectively inhibit trypsin because they contain suitable amino acids in their structures that effectively block the active site of trypsin.

SHARP hydrogel for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a relapsing and remitting inflammatory disease affecting millions of people worldwide. The active phase of IBD is characterized by excessive formation of reactive oxygen species (ROS) in the intestinal mucosa, which further accelerates the inflammatory process. A feasible strategy for the IBD treatment is thus breaking the oxidation-inflammation vicious circle by scavenging excessive ROS with the use of a suitable antioxidant. Herein, we have developed a novel hydrogel system for oral administration utilizing sterically hindered amine-based redox polymer (SHARP) incorporating covalently bound antioxidant SHA groups. SHARP was prepared via free-radical polymerization by covalent crosslinking of 2-hydroxyethyl methacrylate (HEMA), poly(ethylene oxide) methyl ether methacrylate (PEGMA) and a SHA-based monomer, N-(2,2,6,6-tetramethyl-piperidin-4-yl)-methacrylamide. The SHARP hydrogel was resistant to hydrolysis and swelled considerably (∼90% water content) under the simulated gastrointestinal tract (GIT) conditions, and exhibited concentration-dependent antioxidant properties in vitro against different ROS. Further, the SHARP hydrogel was found to be non-genotoxic, non-cytotoxic, non-irritating, and non-absorbable from the gastrointestinal tract. Most importantly, SHARP hydrogel exhibited a statistically significant, dose-dependent therapeutic effect in the mice model of dextran sodium sulfate (DSS)-induced acute colitis. Altogether, the obtained results suggest that the SHARP hydrogel strategy holds a great promise with respect to IBD treatment.

Fluorinated diselenide nanoparticles for radiosensitizing therapy of cancer.

Radiation resistance of cancer cells represents one of the major challenges in cancer treatment. The novel self-assembled fluoralkylated diselenide nanoparticles (fluorosomes) based on seleno-l-cystine (17FSe2) possess redox-active properties that autocatalytically decompose hydrogen peroxide (H2O2) and oxidize the intracellular glutathione (GSH) that results in regulation of cellular oxidative stress. Alkylfluorinated diselenide nanoparticles showed a significant cytotoxic and radiosensitizing effect on cancer cells. The EL-4 tumor-bearing C56BL/6 mice treated with 17FSe2 followed by fractionated radiation treatment (4 × 2Gy) completely suppressed tumor growth. Our results suggest that described diselenide system behaves as a potent radiosensitizer agent targeting tumor growth and preventing tumor recurrence.

Ellipticine-aimed polymer-conjugated auger electron emitter: multistage organelle targeting approach.

Radioactive decay of some radionuclides produces a shower of Auger electrons, potent ionizing radiation within a very short range in living tissue (typically ca. 100 nm). Therefore, they must be brought to DNA-containing cell compartments and preferentially directly to DNA to be fully biologically effective. They may be used for a triple-targeting approach (first targeting, polymer-based system targeting into tumor tissue due to EPR effect; second targeting, pH-controlled release of intercalator-bound Auger electron emitter in slightly acidic tumor tissue or endosome; third targeting, into DNA in cell nucleus by the intercalator) minimizing radiation burden of healthy tissues. We describe a first system of this type, an ellipticine derivative-bound iodine-125 attached to hydrazide moieties containing poly[N-(2-hydroxypropyl)methacrylamide]. The system is stable at pH 7.4 (0% intercalator released after 24 h incubation), while iodine-containing biologically active intercalator is released upon decrease of pH (25% intercalator released after 24 h incubation at pH 5.0-model of late endosomes). Both 2-N-(2-oxobutyl)-9-iodoellipticinium bromide and the noniodinated 2-N-(2-oxobutyl)ellipticinium bromide are potent intercalators, as proven by direct titration with DNA and ethidium displacement assay, and readily penetrate into cell nuclei, as proven by confocal microscopy. They retain chemotherapeutical antiproliferative properties of ellipticine against Raji, EL-4, and 4T1cells with IC(50) in the range 0.27-8.8 µmol/L. Polymer conjugate of 2-N-(2-oxobutyl)-9-iodoellipticinium bromide is internalized into endosomes, releases active drug, possesses cytotoxic activity, and the drug accumulates in cell nuclei.

Biocompatible polypeptide nanogel: Effect of surfactants on nanogelation in inverse miniemulsion, in vivo biodistribution and blood clearance evaluation.

Horseradish peroxidase (HRP)/H2O2-mediated crosslinking of polypeptides in inverse miniemulsion is a promising approach for the development of next-generation biocompatible and biodegradable nanogels. Herein, we present a fundamental investigation of the effects of three surfactants and their different concentrations on the (HRP)/H2O2-mediated nanogelation of poly[N5-(2-hydroxyethyl)-l-glutamine-ran-N5-propargyl-l-glutamine-ran-N5-(6-aminohexyl)-l-glutamine]-ran-N5-[2-(4-hydroxyphenyl)ethyl)-l-glutamine] (PHEG-Tyr) in inverse miniemulsion. The surfactants sorbitan monooleate (SPAN 80), polyoxyethylenesorbitan trioleate (TWEEN 85), and dioctyl sulfosuccinate sodium salt (AOT) were selected and their influence on the nanogel size, size distribution, and morphology was evaluated. The most effective nanogelation stabilization was achieved with 20 wt% nonionic surfactant SPAN 80. The diameter of the hydrogel nanoparticles was 230 nm (dynamic light scattering, DLS) and was confirmed also by nanoparticle tracking analysis (NTA) which showed the diameters ranging from 200 to 300 nm. Microscopy and image analyses showed that the nanogel in the dry state was spherical in shape and had number-average diameter Dn = 26 nm and dispersity Р= 1.91. In the frozen-hydrated state, the nanogel appeared porous and was larger in size with Dn = 182 nm and Р= 1.52. Our results indicated that the nanogelation of the polymer precursor required a higher concentration of surfactant than classical inverse miniemulsion polymerization to ensure effective stabilization. The developed polypeptide nanogel was radiolabeled with 125I, and in vivo biodistribution and blood clearance evaluations were performed. We found that the 125I-labeled nanogel was well-biodistributed in the bloodstream, cleared from mouse blood during 48 h by renal and hepatic pathways and did not provoke any sign of toxic effects.

Enhanced Antitumor Efficacy through an "AND gate" Reactive Oxygen-Species-Dependent pH-Responsive Nanomedicine Approach.

Anticancer drug delivery strategies are designed to take advantage of the differential chemical environment in solid tumors independently, or to high levels of reactive oxygen species (ROS) or to low pH, compared to healthy tissue. Here, the design and thorough characterization of two functionalizable "AND gate" multiresponsive (MR) block amphiphilic copolymers are reported, aimed to take full advantage of the coexistence of two chemical cues-ROS and low pH-present in the tumor microenvironment. The hydrophobic blocks contain masked pH-responsive side chains, which are exposed exclusively in response to ROS. Hence, the hydrophobic polymer side chains will undergo a charge shift in a very relevant pH window present in the extracellular milieu in most solid tumors (pH 5.6-7.2) after demasking by ROS. Doxorubicin (DOX)-loaded nanosized "AND gate" MR polymersomes (MRPs) are fabricated via microfluidic self-assembly. Chemical characterization reveals ROS-dependent pH sensitivity and accelerated DOX release under influence of both ROS and low pH. Treatment of tumor-bearing mice with DOX-loaded nonresponsive and "AND gate" MRPs dramatically decreases cardiac toxicity. The most optimal "AND gate" MRPs outperform free DOX in terms of tumor growth inhibition and survival, shedding light on chemical requirements for successful cancer nanomedicine.