Material characterization of GPX®: A versatile in situ solidifying embolic platform technology.

Endovascular embolization is a minimally invasive procedure during which blood flow to targeted tissues is selectively occluded. The list of clinical indications for embolization continues to expand. Liquid embolic agents are injectable compositions that transition into a solid or semi-solid form when introduced into blood vessels. The mechanism that triggers the liquid-to-solid transition is a key distinguishing feature of liquid embolic agents. GPX is a waterborne liquid embolic agent comprising oppositely charged polyelectrolytes: polyguanidinum and inorganic polyphoshate. In situ solidification is driven by electrostatic condensation of the polyelectrolytes, triggered by ionic strength differentials. We report in vitro characterization of the material properties of GPX, it is in vivo effectiveness in acute animal studies, and its potential for chemoembolization. The viscosity of GPX can be varied over a wide range by adjusting the polyguanidinium MW and/or concentration. Formulation of GPX with either tantalum microparticles (30 wt%) or iodinated radiocontrast agents (300 mgI ml-1) did not significantly change the flow behavior of GPX; the viscosity was independent of shear rate and remained within a clinically practical range (80-160 cP). Formulation of GPX with doxorubicin substantially increased viscosity at low shear rates and resulted in a power law dependence on shear rate. High contrast and effective vascular occlusion were demonstrated in both swine kidneys and rete mirabile. Contrast from iodinated compounds was temporary, dissipating within hours. The doxorubicin in vitro release profile was linear over 90 days. The results demonstrate that GPX is a versatile liquid embolic platform that can be formulated with a wide range of viscosities injectable at clinically practical flow rates, with either transient or permanent contrast, and that can provide prolonged zero-order delivery of doxorubicin to embolized tissues.

Rapid Entrapment of Phenazine Ethosulfate within a Polyelectrolyte Complex on Electrodes for Efficient NAD+ Regeneration in Mediated NAD+-Dependent Bioelectrocatalysis.

Over the past two decades, the designs of redox polymers have become critical to the field of mediated bioelectrocatalysis and are used in commercial glucose biosensors, as well as other bioelectrochemical applications (e.g., energy harvesting). These polymers are specifically used to immobilize redox mediators on electrode surfaces, allowing for self-exchange-based conduction of electrons from enzymes far from the electrode to the electrode surface. However, the synthesis of redox polymers is challenging and results in large batch-to-batch variability. Herein, we report a rapid entrapment of mediators for NAD+-dependent bioelectrocatalysis within reverse ionically condensed polyelectrolytes. A high ionic strength aqueous solution of oppositely charged polyelectrolytes, composed of cationic polyguanidinium (PG) chloride and anionic sodium hexametaphosphate (P6), undergoes phase inversion into a solid microporous polyelectrolyte complex (PEC) when introduced into a low ionic strength aqueous solution. The ionic strength-triggered phase inversion of PGP6 solutions was investigated as a means to entrap mediators on the surface of electrodes for mediated bioelectrocatalysis. Compared to the traditional cross-linked immobilizations using redox polymers, this phase inversion takes place within seconds and requires up to 60 min for complete stabilization. In this work, redox mediator phenazine ethosulfate (PES) was entrapped within PGP6 on electrode surfaces for nicotinamide adenine dinucleotide (NAD+)-dependent bioelectrocatalysis. In the bulk solution, NAD+-dependent dehydrogenase enzymes catalyze the oxidation of the substrate while reducing NAD to reduced nicotinamide adenine dinucleotide (NADH). The resulting NADH is reoxidized to NAD+ by the entrapped PES that gets reduced on the electrode, completing the NAD+-regeneration-based bioelectrocatalysis. To show the use of these new materials in an application, biofuel cells were evaluated using four different anodic enzyme systems (alcohol dehydrogenase, lactate hydrogenase, glycerol dehydrogenase, and glucose dehydrogenase).

Direct bioelectrocatalysis by redox enzymes immobilized in electrostatically condensed oppositely charged polyelectrolyte electrode coatings.

The immobilization of enzymes on an electrode surface is critical in preserving enzyme activity and providing a sufficient electron transfer pathway for bioelectrocatalysis. Here, we present a novel single-step, cross-linker free immobilization for direct bioelectrocatalysis using an ionic strength induced phase inversion of oppositely charged polyelectrolytes. Cationic poly-guanidinyl-propyl-methacrylate (pGPMA, PG) and anionic inorganic polyphosphate, sodium hexametaphosphate (P6) were used to make an electrostatically condensed phase (PGP6). A mixture of PGP6 and laccase (LAC) from Tramates versicolor or HRP (HRP) from Armoracia rusticana were deposited on the electrode surface and were submerged in DI water to form white porous electrode coatings. Each electrode showed a current generation corresponding to the respective substrates via direct bioelectrocatalysis.

Aqueous Liquid-Liquid Phase Separation of Natural and Synthetic Polyguanidiniums.

Protamines are natural polyguanidiniums, arginine(R)-rich proteins involved in the compaction of chromatin during vertebrate spermatogenesis. Salmine, a protamine isolated from salmon sperm, contains 65 mol% R residues, with positively charged guanidino (Gdm⁺) sidechains, and no other amino acids with ionizable or aromatic sidechains. Salmine sulfate solutions undergo liquid-liquid phase separation (LLPS) with a concentration-dependent upper critical solution temperature (UCST). The condensed liquid phase comprises 50 wt % water and >600 mg·ml-¹ salmine with a constant 1:2 ratio of sulfate (SO4²-) to Gdm⁺. Isothermal titration calorimetry, titrating Na2SO4 into salmine chloride above and below the UCST, allowed isolation of exothermic sulfate binding to salmine chloride from subsequent endothermic condensation and exothermic phase separation events. Synthetic random polyacrylate analogs of salmine, with 3-guanidinopropyl sidechains, displayed similar counterion dependent phase behavior, demonstrating that the LLPS of polyguanidiniums does not depend upon subunit sequence or polymer backbone chirality, and was due entirely to Gdm⁺ sidechain interactions. The results provide experimental evidence for like-charge pairing of Gdm⁺ sidechains, and an experimental approach for further characterizing these interactions.

Water-Borne Endovascular Embolics Inspired by the Undersea Adhesive of Marine Sandcastle Worms.

Transcatheter embolization is used to treat vascular malformations and defects, to control bleeding, and to selectively block blood supply to tissues. Liquid embolics are used for small vessel embolization that require distal penetration. Current liquid embolic agents have serious drawbacks, mostly centered around poor handling characteristics and toxicity. In this work, a water-borne in situ setting liquid embolic agent is described that is based on electrostatically condensed, oppositely charged polyelectrolytes-complex coacervates. At high ionic strengths, the embolic coacervates are injectable fluids that can be delivered through long narrow microcatheters. At physiological ionic strength, the embolic coacervates transition into a nonflowing solid morphology. Transcatheter embolization of rabbit renal arteries demonstrated capillary level penetration, homogeneous occlusion, and 100% devascularization of the kidney, without the embolic crossing into venous circulation. The benign water-borne composition and setting mechanism avoids many of the problems of current liquid embolics, and provides precise temporal and spatial control during endovascular embolization.

Sequential combination therapy of ovarian cancer with degradable N-(2-hydroxypropyl)methacrylamide copolymer paclitaxel and gemcitabine conjugates.

For rapid and effective clinical translation, polymer-based anticancer therapeutics need long circulating conjugates that produce a sustained concentration gradient between the vasculature and solid tumor. To this end, we designed second-generation backbone-degradable diblock N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carriers and evaluated sequential combination therapy of HPMA copolymer-paclitaxel and HPMA copolymer-gemcitabine conjugates against A2780 human ovarian carcinoma xenografts. First, extensive in vitro assessment of administration sequence impact on cell cycle, viability, apoptosis, migration, and invasion revealed that treatment with paclitaxel conjugate followed by gemcitabine conjugate was the most effective scheduling strategy. Second, in an in vivo comparison with first-generation (nondegradable, molecular weight below the renal threshold) conjugates and free drugs, the second-generation degradable high-molecular weight conjugates showed distinct advantages, such as favorable pharmacokinetics (three- to five-times half-life compared with the first generation), dramatically enhanced inhibition of tumor growth (complete tumor regression) by paclitaxel and gemcitabine conjugate combination, and absence of adverse effects. In addition, multimodality imaging studies of dual-labeled model conjugates confirmed the efficacy of second-generation conjugates by visualizing more than five-times enhanced tumor accumulation, rapid conjugate internalization, and effective intracellular release of payload. Taken together, the results indicate that the second-generation degradable HPMA copolymer carrier can provide an ideal platform for the delivery of a range of antitumor compounds, which makes it one of the most attractive candidates for potential clinical application.

Cell surface self-assembly of hybrid nanoconjugates via oligonucleotide hybridization induces apoptosis.

Hybrid nanomaterials composed of synthetic and biological building blocks possess high potential for the design of nanomedicines. The use of self-assembling nanomaterials as "bio-mimics" may trigger cellular events and result in new therapeutic effects. Motivated by this rationale, we designed a therapeutic platform that mimics the mechanism of immune effector cells to cross-link surface receptors of target cells and induce apoptosis. This platform was tested against B-cell lymphomas that highly express the surface antigen CD20. Here, two nanoconjugates were synthesized: (1) an anti-CD20 Fab' fragment covalently linked to a single-stranded morpholino oligonucleotide (MORF1), and (2) a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple copies of the complementary oligonucleotide MORF2. We show that the two conjugates self-assemble via MORF1-MORF2 hybridization at the surface of CD20(+) malignant B-cells, which cross-links CD20 antigens and initiates apoptosis. When tested in a murine model of human non-Hodgkin's lymphoma, the two conjugates, either administered consecutively or as a premixture, eradicated cancer cells and produced long-term survivors. The designed therapeutics contains no small-molecule cytotoxic compounds and is immune-independent, aiming to improve over chemotherapy, radiotherapy and immunotherapy. This therapeutic platform can be applied to cross-link any noninternalizing receptor and potentially treat other diseases.

Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer.

Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (-GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer--DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates' in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice.

Efficiency of high molecular weight backbone degradable HPMA copolymer-prostaglandin E1 conjugate in promotion of bone formation in ovariectomized rats.

Multiblock, high molecular weight, linear, backbone degradable HPMA copolymer-prostaglandin E1 (PGE1) conjugate has been synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA), which contains an enzymatically degradable oligopeptide sequence flanked by two dithiobenzoate groups, followed by postpolymerization aminolysis and thiol-ene chain extension. The multiblock conjugate contains Asp8 as the bone targeting moiety and enzymatically degradable bonds in the polymer backbone; in vivo degradation produces cleavage products that are below the renal threshold. Using an ovariectomized (OVX) rat model, the accumulation in bone and efficacy to promote bone formation was evaluated; low molecular weight conjugates served as control. The results indicated a higher accumulation in bone, greater enhancement of bone density, and higher plasma osteocalcin levels for the backbone degradable conjugate.

Synthesis of long-circulating, backbone degradable HPMA copolymer-doxorubicin conjugates and evaluation of molecular-weight-dependent antitumor efficacy.

Backbone degradable, linear, multiblock N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (DOX) conjugates are synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by chain extension via thiol-ene click reaction. The examination of molecular-weight-dependent antitumor activity toward human ovarian A2780/AD carcinoma in nude mice reveals enhanced activity of multiblock, second-generation, higher molecular weight conjugates when compared with traditional HPMA copolymer-DOX conjugates. The examination of body weight changes during treatment indicates the absence of non-specific adverse effects.

Synthesis and evaluation of a backbone biodegradable multiblock HPMA copolymer nanocarrier for the systemic delivery of paclitaxel.

The performance and safety of current antineoplastic agents, particularly water-insoluble drugs, are still far from satisfactory. For example, the currently widely used Cremophor EL®-based paclitaxel (PTX) formulation exhibits pharmacokinetic concerns and severe side effects. Thus, the concept of a biodegradable polymeric drug-delivery system, which can significantly improve therapeutic efficacy and reduce side effects is advocated. The present work aims to develop a new-generation of long-circulating, biodegradable carriers for effective delivery of PTX. First, a multiblock backbone biodegradable N-(2-hydroxypropyl)methacrylamide(HPMA) copolymer-PTX conjugate (mP-PTX) with molecular weight (Mw) of 335 kDa was synthesized by RAFT (reversible addition-fragmentation chain transfer) copolymerization, followed by chain extension. In vitro studies on human ovarian carcinoma A2780 cells were carried out to investigate the cytotoxicity of free PTX, HPMA copolymer-PTX conjugate with Mw of 48 kDa (P-PTX), and mP-PTX. The experiments demonstrated that mP-PTX has a similar cytotoxic effect against A2780 cells as free PTX and P-PTX. To further compare the behavior of this new biodegradable conjugate (mP-PTX) with free PTX and P-PTX in vivo evaluation was performed using female nu/nu mice bearing orthotopic A2780 ovarian tumors. Pharmacokinetics study showed that high Mw mP-PTX was cleared more slowly from the blood than commercial PTX formulation and low Mw P-PTX. SPECT/CT imaging and biodistribution studies demonstrated biodegradability as well as elimination of mP-PTX from the body. The tumors in the mP-PTX treated group grew more slowly than those treated with saline, free PTX, and P-PTX (single dose at 20 mg PTX/kg equivalent). Moreover, mice treated with mP-PTX had no obvious ascites and body-weight loss. Histological analysis indicated that mP-PTX had no toxicity in liver and spleen, but induced massive cell death in the tumor. In summary, this biodegradable drug delivery system has a great potential to improve performance and safety of current antineoplastic agents.

Prostate-cancer-targeted N-(2-hydroxypropyl)methacrylamide copolymer/docetaxel conjugates.

Biodistribution, pharmacokinetics, and efficacy of prostate-cancer-targeted HPMA copolymer/DTX conjugates are evaluated in nude mice bearing prostate cancer C4-2 xenografts. PSMA-specific monoclonal antibodies 3F/11 are used as the targeting moiety. Control conjugates tumor accumulation to total background organs (heart, lung, kidney, liver, spleen and blood) accumulation increase substantially with time for the targeted conjugate, and the ratio at 48 h is 7-fold higher than that at 6 h. Preliminary evaluation of the efficacy of the conjugates in vivo show tumor growth inhibition for all HPMA copolymer/DTX conjugates.

Biodistribution and pharmacokinetic studies of bone-targeting N-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates.

The biodistribution and pharmacokinetics of bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-alendronate conjugates were evaluated following intravenous administration of radioiodinated conjugates to young healthy BALB/c mice. The synthesis of a polymerizable and cathepsin K cleavable alendronate derivative, N-methacryloylglycylglycylprolylnorleucylalendronate, enabled the preparation of HPMA copolymer-alendronate conjugates with varying composition. Using the RAFT (reversible addition-fragmentation chain transfer) polymerization technique, four conjugates with different molecular weight and alendronate content and two control HPMA copolymers (without alendronate) with different molecular weight were prepared. The results of biodistribution studies in mice demonstrated a strong binding capacity of alendronate-targeted HPMA copolymer conjugates to bone. Conjugates with low (1.5 mol%) alendronate content exhibited a similar bone deposition capacity as conjugates containing 8.5 mol % of alendronate. The molecular weight was an important factor in the biodistribution of the HPMA copolymer conjugates. More conjugate structures need to be evaluated, but the data suggest that medium molecular weights (50-100 kDa) might be effective drug carriers for bone delivery.

Release of prostaglandin E(1) from N-(2-hydroxypropyl)methacrylamide copolymer conjugates by bone cells.

Bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-PGE(1) conjugates, containing cathepsin K sensitive spacers, were incubated with induced osteoclasts and osteoblasts, their precursors, and control non-skeletal cells. The release of PGE(1) was monitored by an HPLC assay. In both murine and human cell lines, osteoclasts appeared to be the most active cells in the cleavage (PGE(1) release). Incubation with osteoblasts also resulted in fast PGE(1) release, whereas precursor and control cells released PGE(1) with a substantially slower rate than bone cells (apparently through ester bond cleavage). Experiments in the presence of inhibitors revealed that other enzymes, in addition to cathepsin K, were participating in the cleavage of the conjugate. Confocal fluorescence studies exposed internalization of the conjugate by endocytosis with ultimate localization in the lysosomal/endosomal compartment.

Pharmacokinetic and biodistribution studies of a bone-targeting drug delivery system based on N-(2-hydroxypropyl)methacrylamide copolymers.

Osteotropicity of novel bone-targeted HPMA copolymer conjugates has been demonstrated previously with bone histomorphometric analysis. The pharmacokinetics and biodistribution of this delivery system were investigated in the current study with healthy young BALB/c mice. The 125I-labeled bone-targeted and control (nontargeted) HPMA copolymers were administered intravenously to mice, and their distribution to different organs and tissues was followed using gamma counter and single photon emission computed tomography (SPECT). Both the invasive and noninvasive data further confirmed that the incorporation of D-aspartic acid octapeptide (D-Asp8) as bone-targeting moiety could favorably deposit the HPMA copolymers to the entire skeleton, especially to the high bone turnover sites. To evaluate the influence of molecular weight, three fractions (Mw of 24, 46, and 96 kDa) of HPMA copolymer-D-Asp8 conjugate were prepared and evaluated. Higher molecular weight of the conjugate enhanced the deposition to bone due to the prolonged half-life in circulation, but it weakened the bone selectivity. A higher content of bone-targeting moiety (D-Asp8) in the conjugate is desirable to achieve superior hard tissue selectivity. Further validation of the bone-targeting efficacy of the conjugates in animal models of osteoporosis and other skeletal diseases is needed in the future.

The arthrotropism of macromolecules in adjuvant-induced arthritis rat model: a preliminary study.

PURPOSE: To study the accumulation of macromolecules into the arthritic joints and the possible applications of such phenomenon. METHODS: The accumulation of plasma albumin in the joints of adjuvant-induced arthritis (AIA) rat model was first visualized with Evans blue injection. A N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer contrast agent was then synthesized and injected into the AIA rats to allow qualitative examination of biodistribution and pharmacokinetics of the injected macromolecule with magnetic resonance imaging (MRI). Vital organs and the diseased joints were isolated and examined histologically to correlate with the MRI findings. RESULTS: Deep blue color developed around the arthritic joints of the AIA rat a few hours after the injection of Evans blue. MR imaging of the AIA rats injected with polymer contrast agent demonstrated a gradual but very strong accumulation of the injected polymer in the arthritic joints, which lasted for 1-2 days. Observed differences in the concentration of the injected polymer in the joints correlated with disease severity as assessed histologically. CONCLUSIONS: Profound arthrotropism of macromolecules in the AIA rat model was demonstrated with various imaging tools. These observations should help in the conceptual and practical design of novel macromolecular delivery systems for the imaging and treatment of rheumatoid arthritis.

Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems.

Four polymeric bone-targeting conjugates were synthesized based on poly(ethylene glycol) (PEG, two conjugates) and poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA, two conjugates). The well-known bone-targeting compounds, alendronate and aspartic acid peptide, were used as bone-targeting moieties. Fluorescein isothiocyanate (FITC) was attached to the conjugates as a model drug for detection purposes. The bone-targeting potential of these conjugates was tested in vitro with hydroxyapatite (HA) and in mice. The data obtained indicated that these novel delivery systems could specifically accumulate in the bone tissue.