Glycopolymer Self-Assemblies with Gold(I) Complexed to the Core as a Delivery System for Auranofin

A new glycomonomer <b>1</b> containing a thioacetate group in the anomeric position and mimicking the thiosugar ligand of the gold-based drug auranofin was designed and synthesized in four steps from d-glucose. Both CPADB-mediated homopolymerization and chain extension of a hydrophilic poly­(OEGMEMA) macroRAFT agent were well-controlled with dispersities (<i><i><i>Đ</i></i></i>) below 1.2, highlighting the suitability of thioacetate as a thiol protecting group in RAFT polymerization. Using the homopolymer as a test system, the thioacetate protective groups were selectively removed using hydrazine acetate, and AuPEt₃Cl was subsequently complexed to the exposed thiols to generate a polymeric auranofin analogue with 52% complexation efficiency. Extension of this successful procedure to three block copolymers with differing hydrophobic block lengths, poly­(OEGMEMA)₃₄-<i>b</i>-poly­(<b>1</b>)₄₇, poly­(F-OEGMEMA)₃₂-<i>b</i>-poly­(<b>1</b>)₂₇, and poly­(F-OEGMEMA)₃₂-<i>b</i>-poly­(<b>1</b>)₇ (where “F” in the last two indicates the incorporation of 2 wt % fluorescein methacrylate into the hydrophilic block), produced well-defined complexed block copolymers with complexation efficiencies comparable to that of the homopolymer. Self-assembly of the longest complexed polymer poly­(OEGMEMA)₃₄-<i>b</i>-poly­(<b>1</b>-AuPEt₃)₄₇ generated spherical micelles with a hydrodynamic diameter <i>D</i>_h of 28 nm when prepared by slow water addition to a dilute DMF solution. The IC₅₀ value against OVCAR-3 cells in a serum-free media was 44 μM on a gold concentration basis, compared to 0.3 μM for auranofin itself. The two shorter fluorescent complexed block copolymers formed spherical micelles with <i>D</i>_h 23 and 9 nm, respectively, and proved more cytotoxic than their longer counterpart, both displaying IC₅₀ values of 13.5 μM. The addition of serum to the cell growth medium reduced the cytotoxicity of auranofin by a factor of 3.6 but had a less marked effect on the fluorescent micellar systems, reducing their toxicities by between 2.4 and 2.8 times. These micellar systems therefore show less susceptibility to deactivation by serum proteins (which is the primary limitation to auranofin’s <i>in vivo</i> effectiveness) than the free auranofin, suggesting some protective benefit offered by the hydrophilic shell. Fluorescence microscopy of the two fluorescent systems revealed an accumulation in the lysosomes of the OVCAR-3 cells. The cytotoxicity mechanism may therefore differ from that of auranofin, which is known to interact with mitochondrial proteins

Stabilization of Paclitaxel-Conjugated Micelles by Cross-Linking with Cystamine Compromises the Antitumor Effects against Two- and Three-Dimensional Tumor Cellular Models

Paclitaxel (PTX)-conjugated micelles provide a promising tool for the treatment of prostate cancer. Core cross-linking by incorporating a disulfide bridge is a useful approach to improving the <i>in vivo</i> stability of polymeric micelles. This paper aims to investigate the effects of different degrees of cross-linking on the antitumor efficacy of micelles formed by poly­(ethylene glycol methyl ether acrylate)-<i>b</i>-poly­(carboxyethyl acrylate) (POEGMEA-<i>b</i>-PCEA-PTX) block copolymer. Both two-dimensional (2D) and three-dimensional (3D) <i>in vitro</i> prostate tumor cell models were used to evaluate the un-cross-linked and cross-linked micelles. The cytotoxicity decreased with an increase in the degree of cross-linking upon being tested with 2D cultured cells, and all micelles remained less cytotoxic than free PTX. In the 3D prostate MCTS model, however, there was no statistical difference between the performance of un-cross-linked micelles and free PTX, while increasing cross-linking densities led to significantly relevant decreases in the antitumor efficacy of micelles. These results are contradictory to our previous research using an irreversible cross-linker (1,8-diaminooctane) to stabilize POEGMEA-<i>b</i>-PCEA-PTX conjugate micelles where it was shown that cross-linking accelerates and improves the effects of the micelles when compared to those of un-cross-linked micelles. Further studies that aim to investigate the underlying mechanisms of disulfide bonds when micelles are internalized into cells are desired

Enhanced Delivery of the RAPTA‑C Macromolecular Chemotherapeutic by Conjugation to Degradable Polymeric Micelles

Macromolecular ruthenium complexes are a promising avenue to better and more selective chemotherapeutics. We have previously shown that RAPTA-C [RuCl₂(<i>p</i>-cymene)­(PTA)], with the water-soluble 1,3,5-phosphaadamantane (PTA) ligand, could be attached to a polymer moiety via nucleophilic substitution of an available iodide with an amide in the PTA ligand. To increase the cell uptake of this macromolecule, we designed an amphiphilic block copolymer capable of self-assembling into polymeric micelles. The block copolymer was prepared by ring-opening polymerization of d,l-lactide (3,6-dimethyl-1,4-dioxane-2,5-dione) using a RAFT agent with an additional hydroxyl functionality, followed by the RAFT copolymerization of 2-hydroxyethyl acrylate (HEA) and 2-chloroethyl methacrylate (CEMA). The Finkelstein reaction and reaction with PTA led to polymers that can readily react with the dimer of RuCl₂(<i>p</i>-cymene) to create a macromolecular RAPTA-C drug. RAPTA-C conjugation, micellization, and subsequent cytotoxicity and cell uptake of these polymeric moieties was tested on ovarian cancer A2780, A2780cis, and Ovcar-3 cell lines. Confocal microscopy images confirmed cell uptake of the micelles into the lysosome of the cells, indicative of an endocytic pathway. On average, a 10-fold increase in toxicity was found for the macromolecular drugs when compared to the RAPTA-C molecule. Furthermore, the cell uptake of ruthenium was analyzed and a significant increase was found for the micelles compared to RAPTA-C. Notably, micelles prepared from the polymer containing fewer HEA units had the highest cytotoxicity, the best cell uptake of ruthenium and were highly effective in suppressing the colony-forming ability of cells

Cellular Uptake and Movement in 2D and 3D Multicellular Breast Cancer Models of Fructose-Based Cylindrical Micelles That Is Dependent on the Rod Length

While the shape effect of nanoparticles on cellular uptake has been frequently studied, no consistent conclusions are available currently. The controversy mainly focuses on the cellular uptake of elongated (i.e., filaments or rod-like micelles) as compared to spherical (i.e., micelles and vesicles) nanoparticles. So far, there is no clear trend that proposes the superiority of spherical or nonspherical nanoparticles with conflicting reports available in the literature. One of the reasons is that these few reports available deal with nanoparticles of different shapes, surface chemistries, stabilities, and aspects ratios. Here, we investigated the effect of the aspect ratio of cylindrical micelles on the cellular uptake by breast cancer cell lines MCF-7 and MDA-MB-231. Cylindrical micelles, also coined rod-like micelles, of various length were prepared using fructose-based block copolymers poly­(1-<i>O</i>-methacryloyl-β-d-fructopyranose)-<i>b</i>-poly­(methyl methacrylate). The critical water content, temperature, and stirring rate that trigger the morphological transition from spheres to rods of various aspect ratios were identified, allowing the generation of different kinetically trapping morphologies. High shear force as they are found with high stirring rates was observed to inhibit the formation of long rods. Rod-like micelles with length of 500–2000 nm were subsequently investigated toward their ability to translocate in breast cancer cells and penetrate into MCF-7 multicellular spheroid models. It was found that shorter rods were taken up at a higher rate than longer rods

Superior Chemotherapeutic Benefits from the Ruthenium-Based Anti-Metastatic Drug NAMI‑A through Conjugation to Polymeric Micelles

Macromolecular ruthenium complexes are a promising avenue to better, and more selective, chemotherapeutics. NAMI-A is a ruthenium­(III) drug in Phase II clinical trials that has low cytotoxicity and is inactive against primary tumors. However, it displays both antiangiogenic and anti-invasive properties and has been shown to specifically target tumor metastases, preventing both development and growth. To increase the cytotoxicity and cell uptake of this promising drug, we designed a biocompatible amphiphilic block copolymer capable of self-assembling into polymeric micelles. An appropriate method for the synthesis of a macromolecular NAMI-A drug was identifiedthe polymerization of vinyl imidazole and subsequent addition of a ruthenium­(III) precursor complex. The cytotoxicity of these polymeric moieties was tested on ovarian cancer A2780 and Ovcar-3 and pancreatic AsPC-1 cancer cell lines. On average, across the tested cell lines, a 1.5 times increase in toxicity was found for the NAMI-A copolymer micelles when compared to the NAMI-A molecule. Furthermore, the antimetastatic potential was assessed by evaluating the inhibitory effects on the migration and invasion of cells against three cell lines characterized by differing degrees of malignancy (MDA-MB-231 > MCF-7 > CHO). The NAMI-A micelles were shown to have an improved antimetastatic potential in comparison to NAMI-A

Carbohydrate-Specific Uptake of Fucosylated Polymeric Micelles by Different Cancer Cell Lines

Inspired by upregulated levels of fucosylated proteins on the surfaces of multiple types of cancer cells, micelles carrying β-l-fucose and β-d-glucose were prepared. A range of block copolymers were synthesized by reacting a mixture of 2-azidoethyl β-l-fucopyranoside (FucEtN₃) and 2-azideoethyl β-d-glucopyranoside (GlcEtN₃) with poly­(propargyl methacrylate)-<i>block</i>-poly­(<i>n</i>-butyl acrylate) (PPMA-<i>b</i>-PBA) using copper-catalyzed azide–alkyne cycloaddition (CuAAC). Five block copolymers were obtained ranging from 100 mol % fucose to 100% glucose functionalization. The resulting micelles had hydrodynamic diameters of around 30 nm. In this work, we show that fucosylated micelles reveal an increased uptake by pancreatic, lung, and ovarian carcinoma cell lines, whereas the uptake by the healthy cell lines (CHO) is negligible. This finding suggests that these micelles can be used for targeted drug delivery toward cancer cells

Swollen Micelles for the Preparation of Gated, Squeezable, pH-Responsive Drug Carriers

Natural variations in pH levels of tissues in the body make it an attractive stimuli to trigger drug release from a delivery vehicle. A number of such carriers have been developed but achieving high drug loading combined with low leakage at physiological pH and tunable controlled release at the site of action is an ongoing challenge. Here we report a novel strategy for the synthesis of entirely hydrophilic stimuli-responsive nanocarriers with high passive loading efficiency of doxorubicin (DOX), which show good stability at pH 7 and rapid tunable drug release at intracellular pH. The particles (<i>D</i>_h = 120–150 nm), are prepared by cross-linking the core of swollen micelles of the triblock copolymer poly­[poly­(ethylene glycol) methyl ether methacrylate-<i>b</i>-<i>N</i>,<i>N</i>′-di(methylamino)ethyl methacrylate-<i>b</i>-<i>tert</i>-butyl methacrylate] (poly­(PEGMEM A)<i>-<i>b</i>-</i> PDMAEMA-<i>b</i>-P<i>t</i>BMA)). After subsequent deprotection of the <i>tert</i>-butyl groups a hydrophilic poly­(methacrylic acid) (PMAA) core is revealed. Due to the negative charge in the acidic core the particles absorb 100% of the DOX from solution at pH 7 at up to 50 wt % DOX/polymer, making them extremely simple to load. Unlike other systems, the DMAEMA “gating” shell ensures low drug leakage at pH 7, whereas physical shrinkage of the MAA core allows rapid release below pH 6. The particles deliver DOX with high efficiency to human pancreatic cancer AsPC-1 cell lines, even lowering the IC50 of DOX. As the particles are stable as a dry powder and can be loaded with any mixture of positively charged drugs without complex synthetic or purification steps, we propose they will find use in a range of delivery applications

Direct Polymerization of the Arsenic Drug PENAO to Obtain Nanoparticles with High Thiol-Reactivity and Anti-Cancer Efficiency

PENAO (4-(<i>N</i>-(<i>S</i>-penicillaminylacetyl)­amino) phenylarsonous acid), which is a mitochondria inhibitor that reacts with adenine nucleotide translocator (ANT), is currently being trialed in patients with solid tumors. To increase the stability of the drug, the formation of nanoparticles has been proposed. Herein, the direct synthesis of polymeric micelles based on the anticancer drug PENAO is presented. PENAO is readily available for amidation reaction to form PENAO MA (4-(<i>N</i>-(<i>S</i>-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide) which undergoes RAFT (reversible addition–fragmentation chain transfer) polymerization with poly­(ethylene glycol methyl ether methacrylate) as comonomer and poly­(methyl methacrylate) (pMMA) as chain transfer agent, resulting in p­(MMA)-<i>b</i>-p­(PEG-<i>co</i>-PENAO) block copolymers with 3–15 wt % of PENAO MA. The different block copolymers self-assembled into micelle structures, varying in size and stability (<i>D</i>_h = 84–234 nm, cmc = 0.5–82 μg mol^–1) depending on the hydrophilic to hydrophobic ratio of the polymer blocks and the amount of drug in the corona of the particle. The more stable micelle structures were investigated toward 143B human osteosarcoma cells, showing an enhanced cytotoxicity and cellular uptake compared to the free drug PENAO (IC₅₀ (PENAO) = 2.7 ± 0.3 μM; IC₅₀ (micelle M4) = 0.8 ± 0.02 μM). Furthermore, PENAOs arsonous acid residue remains active when incorporated into a polymer matrix and conjugates to small mono and closely spaced dithiols and is able to actively target the mitochondria, which is PENAO’s main target to introduce growth inhibition in cancer cells. As a result, no cleavable linker between drug and polymer was necessary for the delivery of PENAO to osteosarcoma cells. These findings provide a rationale for <i>in vivo</i> studies of micelle M4 versus PENAO in an osteosarcoma animal model

Polyion Complex Micelle Based on Albumin–Polymer Conjugates: Multifunctional Oligonucleotide Transfection Vectors for Anticancer Chemotherapeutics

Novel biocompatible polyion complex micelles, containing bovine serum albumin (BSA), polymer, and oligonucleotide, were synthesized as a generation of vectors for the gene transfection. Maleimide-terminated poly­((<i>N</i>,<i>N</i>-dimethyl amino) ethyl methacrylate) (PDMAEMA) was prepared via reversible addition–fragmentation chain transfer (RAFT) polymerization and subsequently deprotected. Precise one to one albumin–PDMAEMA bioconjugates have been achieved via 1,4-addition with the free thiol group on Cys34 on the BSA protein. SDS-PAGE and GPC (water) confirmed and quantified the successful conjugation. The conjugation efficiency was found to be independent of the molecular weight of PDMAEMA. After careful pH adjustment, the conjugate could efficiently condense anticancer oligonucleotide, ISIS 5132, which resulted in particles of 15–35 nm with a negative zeta-potential. The size was easily controlled by the polymer chain length. The albumin corona provides complete protection of the cationic polymer and genetic drug, which gave rise to lower potential toxicity from the polymer and higher gene transfection efficiency. Although a control experiment with a traditional PEG-based polyion complex micelle could deliver the drug just as effectively, if not more so, to the ovarian cancer cell line OVCAR-3, this carrier had no selectivity toward cancerous cells and proved just as toxic to HS27 (fibroblast) cell line. In contrast, the albumin-coated particles demonstrated desirable selectivity toward cancerous cells and have been shown to have outstanding performance in the cytotoxicity tests of several carcinoma monolayer cell models. In addition, the complex micelles were able to destroy pancreatic multicellular tumor spheroids, while free ISIS 5132 could not penetrate the spheroid at all. Hence, albumin-coated/oligonucleotide complex micelles are far more promising than the most classical gene delivery vectors

Influencing Selectivity to Cancer Cells with Mixed Nanoparticles Prepared from Albumin–Polymer Conjugates and Block Copolymers

Albumin-based nanoparticles are widely used to delivery anticancer drug because they promote the accumulation of drugs in tumor sites. Nanoparticles with surface immobilized albumin are widely described in literature, although mixed nanoparticles with systematically modified ratios between albumin and PEG-based material are less common. In this work, hybrid nanoparticles were prepared by coassembly of a PEG-based amphiphilic block copolymer together with a polymer–protein conjugate. Poly­(oligo­(ethylene glycol) methyl ether acrylate)–poly­(ε-caprolactone) (POEGMEA–PCL) was prepared by a combination of ring-opening polymerization and reversible addition–fragmentation chain transfer (RAFT) polymerization, while the polymer–protein conjugate was obtained by reacting poly­(ε-caprolactone) with bovine serum albumin (BSA–PCL). Co-assembly of both amphiphiles at different ratios, with and without curcumin as a drug, led to hybrid nanoparticles with various amount of albumin on the particle surface. The resulting hybrid nanoparticles were similar in size (100–120 nm), but increasing the amount of albumin on the surface led to a more-negative ζ potential. The cytotoxicity of the curcumin-loaded nanoparticles was examined on several cell lines. The curcumin-loaded nanoparticles with high amount of albumin led to high cytotoxicity against breast cancer cell lines (MDA-MB-231 and MCF-7), which coincided with high cellular uptake. However, the cytotoxicity of the curcumin-loaded nanoparticles against CHO cells and RAW264.7 cells was reduced, suggesting that albumin can facilitate selectivity toward cancer cells