Toxicity test profile for deep eutectic solvents: A detailed review and future prospects.

Deep eutectic solvents (DESs) are preferable in terms of starting materials, storage and synthesis, simplicity, and component material affordability. In several industries ranging from chemical, electrochemical, biological, biotechnology, material science, etc., DES has demonstrated remarkable potential. Despite all these accomplishments, the safety issue with DES must be adequately addressed. Different DES interacts with the cellular membranes differently. It is not possible to classify all DES as easily biodegradable. By expanding the current understanding of the toxicity and biodegradation of DES, interactions between organisms and cellular membranes can be linked. The DES toxicity profile varies according to their concentration, the nature of the individual components, and how they interact with living things. Therefore, the results of this review can serve as a baseline for DES development in the future.

Nanotechnology for Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM), a grade IV astrocytoma as defined by the World Health Organization (WHO) criteria, is the most common primary central nervous system tumor in adults. After treatment with the current standard of care consisting of surgical resection, concurrent temozolomide (TMZ), and radiation, the median survival is only 15 months. The limited and less-effective treatment options for these highly aggressive GBMs call for the development of new techniques and the improvement of existing technologies. Nanotechnology has shown promise in treating this disease, and some nanomaterials have demonstrated the ability to cross the blood-brain barrier (BBB) and remain in GBM tissues. Although the retention of nanoparticles (NPs) in GBM tissue is necessary to elicit an antitumor response, the delivery of the NP needs to be enhanced. Current research in nanotechnology is directed at increasing the active targeting of GBM tissue not only for the aid of chemotherapeutic drug delivery but also for imaging studies. This review is aimed at describing advancements in increasing nanotechnology specificity to GBM tissue.

The Origin of Ion-Pairing and Redissociation of Ionic Liquid.

We address the possible occurrence of a minimum extent of dissociation (α) of ionic liquid (IL) in IL-solvent mixtures. This phenomenon, known as the redissociation of IL, is responsible for many interesting composition-dependent properties in such mixtures. A thermodynamic model is developed to provide a semiquantitative prediction on the change of α with solvent concentration. It is found that the occurrence of minimum α coincides with the occurrence of a maximum in the mean activity coefficient of dissociated ions, indicating better solvation of free, dissociated ions both with decreasing and increasing solvent concentration. The favorable solvation of free ions is found to change from long-range ion-solvent dielectric polarization to ion-ion-pair dielectric polarization with decreasing solvent concentration. Therefore, the composition dependence of the IL solution dielectric constants, determined from that of the ion-pair and the solvent, is found to be the most important factor for the presence of redissociation in IL solutions.

Reactive oxygen species-activated nanoprodrug of Ibuprofen for targeting traumatic brain injury in mice.

Traumatic brain injury (TBI) is an enormous public health problem, with 1.7 million new cases of TBI recorded annually by the Centers for Disease Control. However, TBI has proven to be an extremely challenging condition to treat. Here, we apply a nanoprodrug strategy in a mouse model of TBI. The novel nanoprodrug contains a derivative of the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen in an emulsion with the antioxidant α-tocopherol. The ibuprofen derivative, Ibu2TEG, contains a tetra ethylene glycol (TEG) spacer consisting of biodegradable ester bonds. The biodegradable ester bonds ensure that the prodrug molecules break down hydrolytically or enzymatically. The drug is labeled with the fluorescent reporter Cy5.5 using nonbiodegradable bonds to 1-octadecanethiol, allowing us to reliably track its accumulation in the brain after TBI. We delivered a moderate injury using a highly reproducible mouse model of closed-skull controlled cortical impact to the parietal region of the cortex, followed by an injection of the nanoprodrug at a dose of 0.2 mg per mouse. The blood brain barrier is known to exhibit increased permeability at the site of injury. We tested for accumulation of the fluorescent drug particles at the site of injury using confocal and bioluminescence imaging of whole brains and brain slices 36 hours after administration. We demonstrated that the drug does accumulate preferentially in the region of injured tissue, likely due to an enhanced permeability and retention (EPR) phenomenon. The use of a nanoprodrug approach to deliver therapeutics in TBI represents a promising potential therapeutic modality.

Reactive oxygen species responsive nanoprodrug to treat intracranial glioblastoma.

Chemotherapy for intracranial gliomas is hampered by limited delivery of therapeutic agents through the blood brain barrier (BBB). An optimal therapeutic agent for brain tumors would selectively cross the BBB, accumulates in the tumor tissue and be activated from an innocuous prodrug within the tumor. Here we show brain tumor-targeted delivery and therapeutic efficacy of a nanometer-sized prodrug (nanoprodrug) of camptothecin (CPT) to treat experimental glioblastoma multiforme (GBM). The CPT nanoprodrug was prepared using spontaneous nanoemulsification of a biodegradable, antioxidant CPT prodrug and α-tocopherol. The oxidized nanoprodrug was activated more efficiently than nonoxidized nanoprodrug, suggesting enhanced therapeutic efficacy in the oxidative tumor microenvironment. The in vitro imaging of U-87 MG glioma cells revealed an efficient intracellular uptake of the nanoprodrug via direct cell membrane penetration rather than via endocytosis. The in vivo study in mice demonstrated that the CPT nanoprodrug passed through the BBB and specifically accumulated in brain tumor tissue, but not in healthy brain tissue and other organs. The accumulation preferably occurred at the periphery of the tumor where cancer cells are most actively proliferating, suggesting optimal therapeutic efficacy of the nanoprodrug. The nanoprodrug was effective in treating subcutaneous and intracranial tumors. The nanoprodrug inhibited subcutaneous tumor growth more than 80% compared with control. The median survival time of mice implanted with an intracranial tumor increased from 40.5 days for control to 72.5 days for CPT nanoprodrug. This nanoprodrug approach is a versatile method for developing therapeutic nanoparticles enabling tumor-specific targeting and treatment. The nontoxic, tumor-specific targeting properties of the nanoprodrug system make it a safe, low cost, and versatile nanocarrier for pharmaceuticals, imaging agents, and diagnostic agents.

The role of long-range interactions in the phase behavior of ionic liquids.

The COSMO-type liquid activity coefficient models are among the most successful methods for the prediction of the phase behaviours of fluids containing ionic liquids. However, these models consider only interactions between species that are in close contact; in other words, the long-range Coulomb interactions, which are known to be important for systems containing charged species, are completely ignored. Here we show that the inclusion of long-range terms (with, e.g., the Pitzer-Debye-Hückel model) is indeed critical to obtain the correct osmotic coefficients and the asymptotic behaviours in the infinite dilution of IL solutions. However, the direct combination of PDH and COSMO-type models does not produce accurate activity coefficient over the whole concentration range because both models contain short range interactions. We show that a recent revision of the COSMO-SAC model, which includes the PDH contributions and a specific treatment of solvent-ion and ion-ion interactions, is capable of providing an accurate description of the properties (phase behaviour, osmotic coefficients) of aqueous IL solutions over the whole concentration range.

Nanoprodrugs of NSAIDs: Preparation and Characterization of Flufenamic Acid Nanoprodrugs.

We demonstrated that hydrophobic derivatives of the nonsteroidal anti-inflammatory drug (NSAID)flufenamic acid (FA), can be formed into stable nanometer-sized prodrugs (nanoprodrugs) that inhibit the growth of glioma cells, suggesting their potential application as anticancer agent. We synthesized highly hydrophobic monomeric and dimeric prodrugs of FA via esterification and prepared nanoprodrugs using spontaneous emulsification mechanism. The nanoprodrugs were in the size range of 120 to 140 nm and physicochemically stable upon long-term storage as aqueous suspension, which is attributed to the strong hydrophobic interaction between prodrug molecules. Importantly, despite the highly hydrophobic nature and water insolubility, nanoprodrugs could be readily activated into the parent drug by porcine liver esterase, presenting a potential new strategy for novel NSAID prodrug design. The nanoprodrug inhibited the growth of U87-MG glioma cells with IC(50) of 20 µM, whereas FA showed IC(50) of 100 µM, suggesting that more efficient drug delivery was achieved with nanoprodrugs.

Oxidative stimuli-responsive nanoprodrug of camptothecin kills glioblastoma cells.

The purpose of this study was to prepare and characterize nanometer-sized prodrug (nanoprodrug) of camptothecin. The camptothecin prodrug was synthesized using tetraethylene glycol spacer linked via carbonate bond to camptothecin and via ester bond to alpha-lipoic acid. The nanoprodrug was prepared through the spontaneous emulsification mechanism using the mixture of camptothecin prodrug and alpha-tocopherol which served as a structural matrix. The nanoprodrug was activated readily by porcine liver esterase and, with a much slower rate, by hydrolytic degradation. Upon longterm storage, the alpha-lipoic acid moiety of the camptothecin prodrug gradually oxidized without loss of structural integrity and therapeutic efficacy. Interestingly, the hydrolytic activation was negligible before the oxidation, but was significantly accelerated after the oxidation of the alpha-lipoic acid moiety, suggesting an oxidative stimuli-responsive activation of the prodrug. The camptothecin nanoprodrug was found to possess significant inhibitory effect on the proliferation of U87-MG glioma cells with an IC(50) of 20 nM.

Chemokine CXC receptor 4--mediated glioma tumor tracking by bone marrow--derived neural progenitor/stem cells.

Malignant gliomas manifest frequent tumor recurrence after surgical resection and/or other treatment because of their nature of invasiveness and dissemination. The recognized brain tumor-tracking property of neural progenitor/stem cells opened the possibility of targeting malignant brain tumors using neural progenitor/stem cells. We and others have previously shown that fetal neural progenitor/stem cells can be used to deliver therapeutic molecules to brain tumors. Our recent work has further shown that gene delivery by bone marrow-derived neural progenitor/stem cells achieves therapeutic effects in a glioma model. In this study, we isolate and characterize bone marrow-derived neural progenitor/stem cells, which also express the chemokine receptor chemokine CXC receptor 4 (CXCR4). We show that CXCR4 is required for their chemotaxis and extracellular matrix invasion against a gradient of glioma soluble factors. Furthermore, beta-galactosidase-labeled bone marrow-derived neural progenitor/stem cells implanted in the contralateral side of the brain were shown to track gliomas as early as day 1 and increased through days 3 and 7. Intracranial glioma tracking by bone marrow-derived neural progenitor/stem cells is significantly inhibited by preincubation of bone marrow-derived neural progenitor/stem cells with a blocking anti-CXCR4 antibody, suggesting a CXCR4-dependent tracking mechanism. Glioma tracking bone marrow-derived neural progenitor/stem cells were found to express progenitor/stem cell markers, as well as CXCR4. Although bromodeoxyuridine incorporation assays and proliferating antigen staining indicated that tumor tracking bone marrow-derived neural progenitor/stem cells were mostly nonproliferating, these cells survive in the local tumor environment with little apoptosis. Elucidating the molecular mechanism of brain tumor tracking by adult source stem cells may provide basis for the development of future targeted therapy for malignant brain tumors.

Preparation and characterization of antioxidant nanospheres from multiple alpha-lipoic acid-containing compounds.

The purpose of this study was to prepare and characterize antioxidant nanospheres composed of multiple alpha-lipoic acid-containing compounds (mALAs). It was found that the nanospheres were remarkably stable under physiologic conditions, maintained the antioxidant property of alpha-lipoic acid, and could be destabilized oxidatively and enzymatically. The preparations were simple and highly reproducible providing a new strategy for the development of nanometer-sized antioxidant biomaterials. The nanospheres may find applications as antioxidant therapeutics and oxidation-responsive antioxidant nanocontainers in drug delivery for pathological conditions characterized by oxidative stress including cancer and neurodegenerative diseases.

Stimuli-responsive antioxidant nanoprodrugs of NSAIDs.

A novel group of alpha-lipoic acid-containing hydrophobic prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) was synthesized and transformed into nanometer-sized prodrugs (nanoprodrugs). Three NSAIDs, indomethacin, ibuprofen and naproxen were linked to alpha-lipoic acid via tetraethylene glycol through hydrolytically degradable ester bonds. The three bifunctional derivatives were dissolved in organic solvents and capable of forming stable nanoprodrugs upon addition of the organic solutions into aqueous phase through the spontaneous emulsification mechanism. Antioxidant property and stimuli-responsiveness of the nanoprodrugs were demonstrated by hypochlorous acid (HOCl) scavenging followed by oxidative destabilization of the nanoprodrugs. The effect of varying NSAIDs on the in vitro hydrolytic prodrug activation catalyzed by porcine liver esterase was investigated by monitoring the rates of NSAIDs hydrolysis from the nanoprodrugs. The remarkable feature of these nanoprodrugs is that despite the highly hydrophobic nature of the derivatives NSAIDs were readily hydrolyzed enzymatically from the nanoprodrugs. Furthermore, the rate of hydrolysis was higher when the nanoprodrugs were oxidized and destabilized upon HOCl scavenging suggesting an enhanced activation of the nanoprodrugs in the oxidative environment.

Nanoconjugate based on polymalic acid for tumor targeting.

A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(beta-l-malic acid) (M(w) 50,000, M(w)/M(n) 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection. Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0-180 min up to 24h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p<0.003). In brain tumors drug accumulated selectively in 24h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal.

Brain tumor tandem targeting using a combination of monoclonal antibodies attached to biopoly(beta-L-malic acid).

Tumor-specific targeting using achievements of nanotechnology is a mainstay of increasing efficacy of anti-tumor drugs. To improve drug targeting we covalently conjugated for the first time two different monoclonal antibodies, an anti-mouse transferrin receptor antibody and a mouse autoimmune anti-nucleosome antibody 2C5, onto the drug delivery nanoplatform, poly(beta-L-malic acid). The active anti-tumor drug components attached to the same carrier molecule were antisense oligonucleotides to vascular protein laminin-8. The resulting drug, a new Polycefin variant, was administered intravenously into glioma-bearing xenogeneic animals. The drug delivery system was targeted across mouse endothelial system by the anti-mouse transferring receptor antibody and to the tumor cell surface by the anti-nucleosome antibody 2C5. The targeting efficacies of the Polycefin variants bearing either two antibodies or each single antibody were compared in vitro and in vivo. ELISA confirmed the co-existence of two antibodies on the same nanoplatform molecule and their functional activities. Fluorescence imaging analysis after 24 h of intravenous injection demonstrated significantly higher tumor accumulation of Polycefin variants with the tandem configuration of antibodies than with single antibodies. The results suggest improved efficacy for tandem configuration of antibodies than for single configurations carried by a drug delivery vehicle.

Inhibition of laminin-8 in vivo using a novel poly(malic acid)-based carrier reduces glioma angiogenesis.

We have previously shown that laminin-8, a vascular basement membrane component, was overexpressed in human glioblastomas multiforme and their adjacent tissues compared to normal brain. Increased laminin-8 correlated with shorter glioblastoma recurrence time and poor patient survival making it a potential marker for glioblastoma diagnostics and prediction of disease outcome. However, laminin-8 therapeutic potential was unknown because the technology of blocking the expression of multi-chain complex proteins was not yet developed. To inhibit the expression of laminin-8 constituents in glioblastoma in vitro and in vivo, we used Polycefin, a bioconjugate drug delivery system based on slime-mold Physarum polycephalum-derived poly(malic acid). It carries an attached transferrin receptor antibody to target tumor cells and to deliver two conjugated morpholino antisense oligonucleotides against laminin-8 alpha4 and beta1 chains. Polycefin efficiently inhibited the expression of both laminin-8 chains by cultured glioblastoma cells. Intracranial Polycefin treatment of human U87MG glioblastoma-bearing nude rats reduced incorporation of both tumor-derived laminin-8 chains into vascular basement membranes. Polycefin was thus able to simultaneously inhibit the expression of two different chains of a complex protein. The treatment also significantly reduced tumor microvessel density (p < 0.001) and area (p < 0.001) and increased animal survival (p < 0.0004). These data suggest that laminin-8 may be important for glioblastoma angiogenesis. Polycefin, a versatile nanoscale drug delivery system, was suitable for in vivo delivery of two antisense oligonucleotides to brain tumor cells causing a reduction of glioblastoma angiogenesis and an increase of animal survival. This system may hold promise for future clinical applications.

Polycefin, a new prototype of a multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery.

A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated beta-poly(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 alpha4 and beta1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.

Stage specific expression of poly(malic acid)-affiliated genes in the life cycle of Physarum polycephalum. Spherulin 3b and polymalatase.

Polymalic acid is receiving interest as a unique biopolymer of the plasmodia of mycetozoa and recently as a biogenic matrix for the synthesis of devices for drug delivery. The acellular slime mold Physarum polycephalum is characterized by two distinctive growth phases: uninucleated amoebae and multinucleated plasmodia. In adverse conditions, plasmodia reversibly transform into spherules. Only plasmodia synthesize poly(malic acid) (PMLA) and PMLA-hydrolase (polymalatase). We have performed suppression subtractive hybridization (SSH) of cDNA from amoebae and plasmodia to identify plasmodium-specific genes involved in PMLA metabolism. We found cDNA encoding a plasmodium-specific, spherulin 3a-like polypeptide, NKA48 (spherulin 3b), but no evidence for a PMLA-synthetase encoding transcript. Inhibitory RNA (RNAi)-induced knockdown of NKA48-cDNA generated a severe reduction in the level of PMLA suggesting that spherulin 3b functioned in regulating the level of PMLA. Unexpectedly, cDNA of polymalatase was not SSH-selected, suggesting its presence also in amoebae. Quantitative PCR then revealed low levels of mRNA in amoebae, high levels in plasmodia, and also low levels in spherules, in agreement with the expression under transcriptional regulation in these cells.

The DNA-polymerase inhibiting activity of poly(beta-l-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum.

The naturally synchronous plasmodia of myxomycetes synthesize poly(beta-l-malic acid), which carries out cell-specific functions. In Physarum polycephalum, poly(beta-l-malate) [the salt form of poly(beta-l-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase alpha of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3H]thymidine 5'-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(beta-l-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(beta-l-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373-380]. To explain the variation in the cell cycle, a periodic competition for poly(beta-l-malate) between DNA polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition they displace DNA polymerase from the complex of poly(beta-l-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(beta-l-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.