Anna Mielańczyk

Fluorescein nanocarriers based on cationic star copolymers with acetal linked sugar cores. Synthesis and biochemical characterization

Well-defined star-shaped copolymers consisting of acetal derivatives of methyl α-D-glucopyranoside cores and polymethacrylate arms containing oxirane pendant groups have been modified with ethylenediamine (EDA) to form amphiphilic and water soluble polycations (positive zeta potential). Normal human dermal fibroblasts (NHDF) and human colon cancer cells (HCT-116) were employed to investigate the cellular uptake and cytotoxicity of diamine-functionalized star copolymers. Star-shaped polymers exhibited low cytotoxicity in NHDF cells, whereas in the case of HCT-116 cell line MTS assay resulted in the decrease of cell proliferation, which was also confirmed by Annexin-V assay indicating the increased rate of HCT-116 cell apoptosis. In the next step, the incorporated amine groups were applied for covalent conjugation of fluorescein isothiocyanate (FITC) with an efficiency of 60–85% estimated by 1H NMR. The additional conjugation experiments were performed by isothermal titration calorimetry (ITC) yielding 45–100% of polymer labeling by fluorescent FITC. The confocal laser scanning microscopy proved cell internalization of the fluorescein-conjugated star copolymers was successful. The model studies showed that this type of star copolymers can be promising carriers for the delivery of drugs.

In Vitro Evaluation of Doxorubicin Conjugates Based on Sugar Core Nonlinear Polymethacrylates toward Anticancer Drug Delivery

V-shaped and star-shaped hydroxylamine-functionalized polymethacrylates designed as nanosized conjugates (<120 nm) with anticancer agent, namely, doxorubicin (DOX), were evaluated in vitro toward their potential usage as drug delivery systems in breast cancer (MCF-7) treatment. Statistical analysis of MTS assay results showed that the 4-arm conjugate (n(DOX) = 16) was the most effective polymeric system against MCF-7/W (wild type) and MCF-7/R (DOX resistant) cell lines. Apoptosis assay analysis showed that MCF-7/R cells cultured with nonlinear copolymers died due to necrosis and late apoptotis, whereas MCF-7/W cells were in early and late apoptosis. Among all tested conjugates, the most promising results with induction of apoptosis without inducing necrosis in both MCF-7 cell lines were obtained for conjugate based on 4-arm stars with low content of DOX. The cell cycle assay revealed that increase of MMA units in 4-arm copolymers induced MCF-7/R cell arrest in the SubG1 phase. In the same cell line, the corresponding conjugates triggered S and G2/M arrest. Gradual internalization of the chosen conjugate by MCF-7/R cells was monitored via fluorescence microscopy showing its main localization in the cytoplasm.

Designing Drug Conjugates Based on Sugar Decorated V-Shape and Star Polymethacrylates: Influence of Composition and Architecture of Polymeric Carrier.

Amphiphilic ethylenediamine (EDA)-functionalized V-shape and star copolymers with centrally placed methyl-α,D-glucopyranoside were designed as nanocarriers. Anticancer doxorubicin (DOX) was conjugated in water via amine groups in copolymers to form ketimine linkers. Variations of arm length and number (40-65 units per arm and 2 vs 3 vs 4 arms), DOX feed amount, and conjugation site content (50-160 units of EDA groups), as responsible for efficiency of drug attachment (10-60 units of conjugated DOX) and its release at various pH (5.0 vs 7.4), were studied to demonstrate potential for drug delivery. Size of conjugate particles (10-195 nm) formed in aqueous solution was strongly dependent on the polymer composition and topology. The broad range of drug amounts (25-95%) were detected by the precipitation method, showing pH sensitivity by some polymeric conjugates with faster DOX release in acidic conditions.

Design of systems based on 4-armed star-shaped polyacids for indomethacin delivery

The 4-armed star-shaped copolymers containing methyl (meth)acrylate (MMA, MA) and (meth)acrylic acid (MAA, AA) units were synthesized by ATRP with the use of pentaerythritol (PTL) derivatives as tetrafunctional initiators and by post-polymerization modification to deprotect acidic units. The resultant MMA/MAA, MMA/AA, and MA/MAA based stars were varied by composition of arms using various initial proportions of comonomer pairs. The amphiphilic copolymers were self-assembled by dialysis or solvent evaporation methods into particles, which were also loaded with indomethacin (IMC). The sizes of polymeric aggregates were in the range of 100–200 nm, whereas drug loading efficiency was 10–85%. The in vitro release of IMC in phosphate buffer solution (PBS) demonstrated a higher rate of drug release at pH 5.0 than at 7.4. Drug release profiles can be adjusted by the content of the hydrophilic fraction (15–98%) and distribution of acidic units among arm chains (statistical vs. gradient vs. reverse gradient) as indicated by the broad range of the maximum drug release (10–85%).

Miktoarm star copolymers from D-(−)-salicin core aggregated into dandelion-like structures as anticancer drug delivery systems: synthesis, self-assembly and drug release

The β-glucoside-based heterofunctional initiator was used in the synthesis of well-defined eight-armed miktopolymers by sequential ring opening polymerization (ROP) of ε-caprolactone (CL) and atom transfer radical (co)polymerization (ATRP) of methyl methacrylate (MMA) and/or tert-butyl methacrylate (tBMA). Consequently, methacrylic acid (MAA) repeating units were introduced via selective cleavage of pendant tert-butyl protecting groups. Both the amphiphilic copolymers and miktoarm copolymers were self-assembled at 37°C and pH 7.4. The aggregates of miktoarm polymers were larger than that formed by polymethacrylate homoarm stars (≥250nm vs ≤200nm). The critical aggregation concentrations (CAC) of (mikto)stars were relatively low (0.006-0.411mg/mL) and decreased with the increase in MAA fraction content. Both MAA-based mikto- and homoarmed (co)polymers with shorter arms exhibited lower doxorubicin (DOX) loading capacity, whereas camptothecin (CPT) was encapsulated preferably by miktostars. The kinetic profiles of drug release showed that the rate of release was higher at acidic environment (pH 5.0) than in neutral pH. In the most cases the studied miktopolymer systems demonstrated the well-controlled delivery of the model anticancer drugs, which can be adjusted by structural parameters of polymeric carriers.

Cellular response to star-shaped polyacids. Solution behavior and conjugation advantages

The nanosized (∼10nm in 0.01M PBS and 210nm in water) star-shaped polymethacrylates with various content of pendant carboxyl groups were characterized via basic physicochemical and biological properties toward their use as drug carriers for intravenous administration. The carboxyl groups in polymer were employed to conjugate fluorescein (FA) or doxorubicin (DOX) via amide bond formation. In case of DOX, the conjugation efficiency was higher (4.0-16.0%) than of FA conjugation (1.5-4.5%) for corresponding copolymers. The solubility of conjugates strongly depended on the type of attached compound, that is free carriers and their FA conjugates were water-soluble, whereas DOX conjugates were insoluble in water. Cytotoxicity tests performed on model fibroblast and epithelial cell lines showed that negatively charged copolymers (ZP ranged from -75 to -25mV) were slightly toxic for normal cells (NHDF) and non-toxic for cancer cell lines (HCT-116 and MCF-7/R). The copolymer dose equal to 125μg/mL resulted in cell viability 118% towards NHDF and 90% for HCT116 cells. The internalization of a representative polymer-fluorescein conjugate by HCT-116 and its accumulation in cytoplasm was proven via fluorescence microscopy. MMA/MAA stars showed no adverse effect on HCT-116 cells, hence fluorescein-tagged polymers might be applied as fluorescence probes for in vitro imaging, whereas doxorubicin-tagged polymers might be developed as a new polymeric drug carriers.