Multifunctional decoration of alpha-tocopheryl succinate-based NP for cancer treatment: effect of TPP and LTVSPWY peptide.

Active targeting not only of a specific cell but also a specific organelle maximizes the therapeutic activity minimizing adverse side effects in healthy tissues. The present work describes the synthesis, characterization, and in vitro biological activity of active targeting nanoparticles (NP) for cancer therapy based on α-tocopheryl succinate (α-TOS), a well-known mitocan, that selectively induces apoptosis of cancer cells and proliferalting endothelial cells. Human epidermal growth factor receptor 2 (HER2) targeting peptide LTVSPWY (PEP) and triphenylphosphonium lipophilic cation (TPP) were conjugated to a previously optimized RAFT block copolymer that formed self-assembled NP of appropriate size for this application and low polydispersity by self-organized precipitation method. PEP and TPP were included in order to target not only HER2 positive cancer cells, but also the mitochondria of these cancer cells, respectively. The in vitro experiments demonstrated the faster incorporation of the active-targeting NP and the higher accumulation of TPP-bearing NP in the mitochondria of MDA-MB-453 HER2 positive cancer cells compared to non-decorated NP. Moreover, the encapsulation of additional α-TOS in the hydrophobic core of the NP was achieved with high efficiencies. The loaded NP presented higher cytotoxicity than unloaded NP but preserved their selectivity against cancer cells in a range of tested concentrations.

Photothermal and photodynamic activity of polymeric nanoparticles based on α-tocopheryl succinate-RAFT block copolymers conjugated to IR-780.

The aim of this work was the generation of a multifunctional nanopolymeric system that incorporates IR-780 dye, a near-infrared (NIR) imaging probe that exhibits photothermal and photodynamic properties; and a derivate of α-tocopheryl succinate (α-TOS), a mitochondria-targeted anticancer compound. IR-780 was conjugated to the hydrophilic segment of copolymer PEG-b-polyMTOS, based on poly(ethylene glycol) (PEG) and a methacrylic derivative of α-tocopheryl succinate (MTOS), to generate IR-NP, self-assembled nanoparticles (NPs) in aqueous media which exhibit a hydrophilic shell and a hydrophobic core. During assembly, the hydrophobic core of IR-NP could encapsulate additional IR-780 to generate derived subspecies carrying different amount of probe (IR-NP-eIR). Evaluation of photo-inducible properties of IR-NP and IR-NP-eIR were thoroughly assessed in vitro. Developed nanotheranostic particles showed distinct fluorescence and photothermal behavior after excitation by a laser light emitting at 808nm. Treatment of MDA-MB-453 cells with IR-NP or IR-NP-eIR resulted in an efficient internalization of the IR-780 dye, while subsequent NIR-laser irradiation led to a severe decrease in cell viability. Photocytoxicity conducted by IR-NP, which could not be attributed to the generation of lethal hyperthermia, responded to an increase in the levels of intracellular reactive oxygen species (ROS). Therefore, the fluorescence imaging and inducible phototoxicity capabilities of NPs derived from IR-780-PEG-b-polyMTOS copolymer confer high value to these nanotheranostics tools in clinical cancer research. STATEMENT OF SIGNIFICANCE: Multifunctional polymeric nanoparticles (NPs) that combine imaging and therapeutic properties are highly valuable in cancer treatment. In this paper we describe the development of NPs that are fluorescent in the near-infrared (NIR). This is important for their visualization in living tissues that present low absorption and low autofluorescence in this wavelength region (between 700 and 1000nm). Moreover, NPs present photothermal and photodynamic properties when NIR irradiated: the NPs produce an efficient increment of temperature and increase the intracellular reactive oxygen species (ROS) when laser irradiated at 808nm. These tuneable photoinduced properties make the NPs highly cytotoxic after NIR irradiation and provide a new tool for highly precise cancer treatment.

Enhanced Bioactivity of α-Tocopheryl Succinate Based Block Copolymer Nanoparticles by Reduced Hydrophobicity.

Well-structured amphiphilic copolymers are necessary to obtain self-assembled nanoparticles (NPs) based on synthetic polymers. Highly homogeneous and monodispersed macromolecules obtained by controlled polymerization have successfully been used for this purpose. However, disaggregation of the organized macromolecules is desired when a bioactive element, such as α-tocopheryl succinate, is introduced in self-assembled NPs and this element must be exposed or released to exert its action. The aim of this work is to demonstrate that the bioactivity of synthetic NPs based on defined reversible addition-fragmentation chain transfer polymerization copolymers can be enhanced by the introduction of hydrophilic comonomers in the hydrophobic segment. The amphiphilic terpolymers are based on poly(ethylene glycol) (PEG) as hydrophilic block, and a hydrophobic block based on a methacrylic derivative of α-tocopheryl succinate (MTOS) and small amounts of 2-hydroxyethyl methacrylate (HEMA) (PEG-b-poly(MTOS-co-HEMA)). The introduction of HEMA reduces hydrophobicity and introduces "disorder" both in the homogeneous blocks and the compact core of the corresponding NPs. These NPs are able to encapsulate additional α-tocopheryl succinate (α-TOS) with high efficiency and their biological activity is much higher than that described for the unmodified copolymers, proposedly due to more efficient degradation and release of α-TOS, demonstrating the importance of the hydrophilic-hydrophobic balance.

α-Tocopheryl succinate-based amphiphilic block copolymers obtained by RAFT and their nanoparticles for the treatment of cancer.

α-Tocopheryl succinate (α-TOS) is a well-known mitochondrially targeted anticancer compound. However, the major factor limiting the use of α-TOS is its low solubility in physiological media. To overcome this problem, the aim of this work is the preparation of new polymeric and active α-TOS-based nanovehicle with a precise control over its macromolecular architecture. Reversible addition-fragmentation chain transfer polymerization (RAFT) is used to synthesize an α-TOS amphiphilic block copolymer with highly homogeneous molecular weight and relatively narrow dispersity. Macro-chain transfer agents (macro-CTA) based on poly(ethylene glycol) (PEG) of different molecular weights (MW, ranging from 4.6 to 20 kDa) are used to obtain block copolymers with different hydrophilic/hydrophobic ratios with PEG being the hydrophilic block and a methacrylic derivative of α-tocopheryl succinate (MTOS) being the monomer that formed the hydrophobic block. PEG-b-poly(MTOS) form spherical nanoparticles (NPs) by self-organized precipitation (SORP) or solvent exchange in aqueous media enabling to encapsulate and deliver hydrophobic molecules in their core. The resulting NPs are rapidly endocytosed by cancer cells. The biological activity of the synthesized NPs are found to depend on the MW of PEG, with NP comprised of the higher MW copolymer resulting in the lower bioactivity due to PEG shielding inhibiting cellular uptake by endocytosis. Moreover, the biological activity also depends on the MTOS content, as the biological activity increases as a function of MTOS concentration.

Anticancer and antiangiogenic activity of surfactant-free nanoparticles based on self-assembled polymeric derivatives of vitamin E: structure-activity relationship.

α-Tocopheryl succinate (α-TOS) is a well-known mitochondrially targeted anticancer compound, however, it is highly hydrophobic and toxic. In order to improve its activity and reduce its toxicity, new surfactant-free biologically active nanoparticles (NP) were synthesized. A methacrylic derivative of α-TOS (MTOS) was prepared and incorporated in amphiphilic pseudoblock copolymers when copolymerized with N-vinylpyrrolidone (VP) by free radical polymerization (poly(VP-co-MTOS)). The selected poly(VP-co-MTOS) copolymers formed surfactant-free NP by nanoprecipitation with sizes between 96 and 220 nm and narrow size distribution, and the in vitro biological activity was tested. In order to understand the structure-activity relationship three other methacrylic monomers were synthesized and characterized: MVE did not have the succinate group, SPHY did not have the chromanol ring, and MPHY did not have both the succinate group and the chromanol ring. The corresponding families of copolymers (poly(VP-co-MVE), poly(VP-co-SPHY), and poly(VP-co-MPHY)) were synthesized and characterized, and their biological activity was compared to poly(VP-co-MTOS). Both poly(VP-co-MTOS) and poly(VP-co-MVE) presented triple action: reduced cell viability of cancer cells with little or no harm to normal cells (anticancer), reduced viability of proliferating endothelial cells with little or no harm to quiescent endothelial cells (antiangiogenic), and efficiently encapsulated hydrophobic molecules (nanocarrier). The anticancer and antiangiogenic activity of the synthesized copolymers is demonstrated as the active compound (vitamin E or α-tocopheryl succinate) do not need to be cleaved to trigger the biological action targeting ubiquinone binding sites of complex II. Poly(VP-co-SPHY) and poly(VP-co-MPHY) also formed surfactant-free NP that were also endocyted by the assayed cells; however, these NP did not selectively reduce cell viability of cancer cells. Therefore, the chromanol ring of the vitamin E analogues has an important role in the biological activity of the copolymers. Moreover, when succinate moiety is substituted and vitamin E is directly linked to the macromolecular chain through an ester bond, the biological activity is maintained.

PHACOS, a functionalized bacterial polyester with bactericidal activity against methicillin-resistant Staphylococcus aureus.

Biomaterial-associated infections represent a significant clinical problem, and treatment of these microbial infections is becoming troublesome due to the increasing number of antibiotic-resistant strains. Here, we report a naturally functionalized bacterial polyhydroxyalkanoate (PHACOS) with antibacterial properties. We demonstrate that PHACOS selectively and efficiently inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) both in vitro and in vivo. This ability has been ascribed to the functionalized side chains containing thioester groups. Significantly less (3.2-fold) biofilm formation of S. aureus was detected on PHACOS compared to biofilms formed on control poly(3-hydroxyoctanoate-co-hydroxyhexanoate) and poly(ethylene terephthalate), but no differences were observed in bacterial adhesion among these polymers. PHACOS elicited minimal cytotoxic and inflammatory effects on murine macrophages and supported normal fibroblast adhesion. In vivo fluorescence imaging demonstrated minimal inflammation and excellent antibacterial activity for PHACOS compared to controls in an in vivo model of implant-associated infection. Additionally, reductions in neutrophils and macrophages in the vicinity of sterile PHACOS compared to sterile PHO implant were observed by immunohistochemistry. Moreover, a similar percentage of inflammatory cells was found in the tissue surrounding sterile PHACOS and S. aureus pre-colonized PHACOS implants, and these levels were significantly lower than S. aureus pre-colonized control polymers. These findings support a contact active surface mode of antibacterial action for PHACOS and establish this functionalized polyhydroxyalkanoate as an infection-resistant biomaterial.