Darin Y. Furgeson

Toxicity Assessments of Multisized Gold and Silver Nanoparticles in Zebrafish Embryos

The potential toxicity of nanoparticles is addressed by utilizing a putative attractive model in developmental biology and genetics: the zebrafish (Danio rerio). Transparent zebrafish embryos, possessing a high degree of homology to the human genome, offer an economically feasible, medium-throughput screening platform for noninvasive real-time assessments of toxicity. Using colloidal silver (cAg) and gold nanoparticles (cAu) in a panoply of sizes (3, 10, 50, and 100 nm) and a semiquantitative scoring system, it is found that cAg produces almost 100% mortality at 120 h post-fertilization, while cAu produces less than 3% mortality at the same time point. Furthermore, while cAu induces minimal sublethal toxic effects, cAg treatments generate a variety of embryonic morphological malformations. Both cAg and cAu are taken up by the embryos and control experiments, suggesting that cAg toxicity is caused by the nanoparticles themselves or Ag(+) that is formed during in vivo nanoparticle destabilization. Although cAg toxicity is slightly size dependent at certain concentrations and time points, the most striking result is that parallel sizes of cAg and cAu induce significantly different toxic profiles, with the former being toxic and the latter being inert in all exposed sizes. Therefore, it is proposed that nanoparticle chemistry is as, if not more, important than specific nanosizes at inducing toxicity in vivo. Ultimately such assessments using the zebrafish embryo model should lead to the identification of nanomaterial characteristics that afford minimal or no toxicity and guide more rational designs of materials on the nanoscale.

Zebrafish as a correlative and predictive model for assessing biomaterial nanotoxicity.

The lack of correlative and predictive models to assess acute and chronic toxicities limits the rapid pre-clinical development of new therapeutics. This barrier is due in part to the exponential growth of nanotechnology and nanotherapeutics, coupled with the lack of rigorous and robust screening assays and putative standards. It is a fairly simple and cost-effective process to initially screen the toxicity of a nanomaterial by using invitro cell cultures; unfortunately it is nearly impossible to imitate a complimentary invivo system. Small mammalian models are the most common method used to assess possible toxicities and biodistribution of nanomaterials in humans. Alternatively, Daniorerio, commonly known as zebrafish, are proving to be a quick, cheap, and facile model to conservatively assess toxicity of nanomaterials.

Structural optimization of a “smart” doxorubicin–polypeptide conjugate for thermally targeted delivery to solid tumors

A thermoresponsive, genetically engineered, elastin-like polypeptide (ELP) containing a C-terminal cysteine residue was synthesized and purified by inverse transition cycling (ITC) and conjugated to doxorubicin (Dox) molecules through four different pH-sensitive, maleimide-activated, hydrazone linkers. The efficiency of Dox activation, conjugation ratios to ELP and biophysical characterization-hydrodynamic radius (Rh) and the temperature transition kinetics-of the ELP-Dox conjugates and pH-mediated release of Dox were quantified in this study. Conjugation ratios of the maleimide-activated Dox to the thiol group of a unique cysteine in the ELP were close to unity. The Rh of the conjugate increased as the linker length between the ELP backbone and Dox was increased. The linker structure and length had little effect on the Tt of the ELP-Dox conjugates, as all conjugates exhibited Tts that were similar to the native ELP. However, the ELP-Dox conjugates with longer linkers exhibited slower transition kinetics compared to the ELP-Dox conjugates with shorter linkers. The highest release of the ELP-Dox conjugate by cleavage of the hydrazone bond at pH 4 was nearly 80% over 72 h and was exhibited by the conjugate with the shortest linker.

Folate-PEG-folate-graft-polyethylenimine-based gene delivery.

Folate-polyethylene glycol-folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized by linking folic acid to both ends of a mono-functional PEG and then grafting to PEL The graft ratio was determined using Beers law by measuring the UV absorbance at 363 nm. The pH profile, diameter and shape of the carriers were determined. Transfection efficiencies were optimized in normal smooth muscle cells (SMC) and CT-26 colon adenocarcinoma cells using plasmid DNA encoding luciferase reporter gene. Free folic acid was shown to inhibit transfection with FPF-2.3g-PEI at neutral charge ratio. Relative toxicity between PEI and the modified carrier was measured using MTT colorimetric assay. Therapeutic potential of pm1FN-γ complexed with these polymeric carriers in terms of gene expression was determined at protein and mRNA levels using ELISA and RT-PCR. FPF-g-PEI was determined to have 2.3 folate-PEG-folate (FPF) linear polymers grafted to each PEI molecule. The average molecular weight was measured to be ~33,500 Mw and the pH profile was characteristic of endosomal disruption capacity. Atomic Force Microscopy (AFM) and Dynamic Laser Light Scattering (DLLS) indicated FPF-2.3g-PEI and PEI (at 2 w/w ratio) efficiently condensed plasmid DNA resulting in oblique spheroid polyplexes with a mean diameter of ~150 nm. FPF-2.3g-PEI was superior to PEI in terms of cytotoxicity and transfection efficiency in cancer cells. Smooth muscle cells showed no specificity for folate tethered complexes, where PEI/pLuc complexes yielded higher efficiencies.

Novel water insoluble lipoparticulates for gene delivery

AbstractPurpose. The objective was to design and prepare water insoluble lipoparticulates (ISLPs) for efficient gene delivery to lung tissue. Methods. Nona{(ethylenimine)-co-[(2-aminoethyl)-N-choleseteryl-oxycarbonyl-ethylenimine]} (NEACE-T) was synthesized in both its free-base and chloride salt-forms using linear polyethylenimine (PEI, Mw 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitele- chelic Nα-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by 1H-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a secondary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a colipid, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1/1, 1/2, and 2/1 molar ratios and the lipoparticulates were hydrated and filtered. ISLP/p2CMVmIL-12 complexes were characterized for particle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Results. Transgene expression was dependent on the site of cholesterol conjugation, lipononamer:colipid molar ratio, and ISLP/p2CMVmIL-12 charge ratios. ISLP/p2CMVmIL-12 complexes were nontoxic to murine colon adenocarcinoma (CT-26) cells at 9/1 (±) or lower, had a mean particle size of 330-400 nm while the ζ potential varied from 36-39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size comparable to that determined by dynamic light scattering. ISLP/p2CMVmIL-12 complexes prepared using free-base NEACE-T:DOPE (1/2) at charge ratios of 3/1 and 5/1 (±) provided the highest levels of transgene expression, 18 times more than the levels provided by the salt-form. Secreted levels of mIL-12 p70 were 75 times higher for ISLP/p2CMVmIL-12 complexes than naked p2CMVmIL-12 and nearly 4 times higher than PEI 25 kDa/p2CMVmIL-12 complexes. Conclusions. The transfection efficiency of the ISLPs was dependent on the site of cholesterol conjugation, amount of colipid, and charge ratio. The highest levels of transgene expression were provided by NEACE-T:DOPE (1/2)/p2CMVmIL-12 at a 3/1 (±) charge ratio.

A myristoylated cell-penetrating peptide bearing a transferrin receptor-targeting sequence for neuro-targeted siRNA delivery.

Many neurodegenerative disorders (NDDs) are characterized by aggregation of aberrant proteins and extensive oxidative stress in brain cells. As a treatment option for NDDs, RNA interference (RNAi) is a promising approach to suppress the activation of abnormal genes and negative regulators of antioxidant genes. Efficient neuro-targeted siRNA delivery requires a delicate optimization of nucleic acid carriers, quite distinct from putative pDNA carriers in regard to stable condensation and serum protection of siRNA, blood–brain barrier (BBB) bypass, effective siRNA delivery to brain cells, and functional release of bioactive siRNA at therapeutic levels. Here, we propose that a myristic acid conjugated, cell-penetrating peptide (transportan; TP), equipped with a transferrin receptor-targeting peptide (myr-TP-Tf), will lead to stable encapsulation of siRNA and targeted delivery of siRNA to brain cells overcoming the BBB. Myr-TP-Tf was successfully prepared by solid-phase peptide synthesis with high purity. Myr-TP-Tf–siRNA complexes formulated at a 20:1 (peptide–siRNA) molar ratio provided prolonged siRNA stability against serum and ribonuclease treatment. Fluorescence images clearly indicated that siRNA uptake was successfully achieved by myr-TP-Tf complexes in both a murine brain endothelioma and a human glioma cell line. The luciferase assay and the human placental alkaline phosphatase (hPAP) reporter assay results demonstrated the functional gene silencing effect of myr-TP-Tf–siRNA complexes in a human glioma cell line as well as in primary murine neurons/astrocytes, supportive of successful release of bioactive siRNA into the cytosol. Finally, the transcytosis assay revealed that favorable siRNA transport via receptor-mediated transcytosis was mediated by myr-TP-Tf complexes. In summary, these data suggest that myr-TP-Tf peptides possess promising properties as a vehicle for neuro-targeted siRNA delivery. We will further study this peptide in vitro and in vivo for transport mechanism kinetics and to validate its capability to deliver siRNA to the brain, respectively.

The Primacy of Physicochemical Characterization of Nanomaterials for Reliable Toxicity Assessment: A Review of the Zebrafish Nanotoxicology Model

Engineered nanomaterials (ENMs) have become increasingly prevalent in the past two decades in academic, medical, commercial, and industrial settings. The unique properties imbued with nanoparticles, as the physiochemical properties change from the bulk material to the surface atoms, present unique and often challenging characteristics that larger macromolecules do not possess. While nanoparticle characteristics are indeed exciting for unique chemistries, surface properties, and diverse applications, reports of toxicity and environmental impacts have tempered this enthusiasm and given cause for an exponential increase for concomitant nanotoxicology assessment. Currently, nanotoxicology is a steadily growing with new literature and studies being published more frequently than ever before; however, the literature reveals clear, inconsistent trends in nanotoxicological assessment. At the heart of this issue are several key problems including the lack of validated testing protocols and models, further compounded by inadequate physicochemical characterization of the nanomaterials in question and the seminal feedback loop of chemistry to biology back to chemistry. Zebrafish (Danio rerio) are emerging as a strong nanotoxicity model of choice for ease of use, optical transparency, cost, and high degree of genomic homology to humans. This review attempts to amass all contemporary nanotoxicology studies done with the zebrafish and present as much relevant information on physicochemical characteristics as possible. While this report is primarily a physicochemical summary of nanotoxicity studies, we wish to strongly emphasize that for the proper evolution of nanotoxicology, there must be a strong marriage between the physical and biological sciences. More often than not, nanotoxicology studies are reported by groups dominated by one discipline or the other. Regardless of the starting point, nanotoxicology must be seen as an iterative process between chemistry and biology. It is our sincere hope that the future will introduce a paradigm shift in the approach to nanotoxicology with multidisciplinary groups for data analysis to produce predictive and correlative models for the end goal of rapid preclinical development of new therapeutics into the clinic or insertion into environmental protection.

Thermo-targeted drug delivery of geldanamycin to hyperthermic tumor margins with diblock elastin-based biopolymers

The tumor margins are the barrier to hepatocellular carcinoma (HCC) eradication for tumors>3 cm. Indeed, inadequately treated tumor margins commonly result in local and regional HCC recurrence with increased size and mass. Tumor recurrence is a common problem with chemotherapy, radiotherapy, thermal ablation, and/or surgical resection, by the inability to properly treat the tumor core and the tumor margins. Here we present novel thermosensitive biopolymer-drug conjugates for thermo-targeted chemotherapy at hyperthermic isotherms produced by focal, locoregional thermal ablation. The chemotherapeutic target is heat shock protein 90 (HSP90), a key molecular chaperone of several, and potent pro-oncogenic pathways including Akt, Raf-1, and mutated p53 that is upregulated in HCC. To inhibit HSP90, we have chosen geldanamycin (GA), a potent HSP90 inhibitor. GA has gained significant attention for its low IC50 ~ 1 nM and inhibition of Akt and Raf-1, amongst other critical pro-oncogenic pathways. Despite such evidence, clinical trials of GA have not shown promise due to off-target toxicity and poor formulation design. Here, we propose using diblock elastin-based biopolymers as a Ringsdorf macromolecular GA solubilizer--a new generation containing functional poly(Asp)/(Glu) blocks for facile drug conjugation and an ELP block for thermo-targeting of hyperthermic ablative margins. GA release is controlled by pH-sensitive, covalent hydrazone bonds with the biopolymer backbone to avoid systemic toxicity and off-target effects. The resultant biopolymer-conjugates form stable nanoconstructs and display tunable, acute phase transitions at high temperatures. Drug release kinetics are favorable with or without the presence of serum. Thermo-targeted chemotherapy and synchronous thermal ablation provide a unique opportunity for simultaneous destruction of the HCC ablative margins and tumor core for focal, locoregional control of HCC.

Biodegradable hybrid recombinant block copolymers for non-viral gene transfection

Thermal targeting of therapeutic genes can enhance local gene concentration to maximize their efficacy. However, lack of safe and efficient carriers has impeded the development of this delivery option. Herein, we report the preparation and evaluation of a hybrid recombinant material, p[Asp(DET)](53)ELP(1-90), that possess a thermo-responsive elastin-like polypeptide (ELP) segment and a diethylenetriamine (DET) modified poly-L-aspartic acid segment. The term, hybrid, indicates that the material was prepared by genetic engineering and synthetic chemistry. In summary, the thermal phase transition behavior and cytotoxicity of the biodegradable copolymer were studied. The polyplexes formed by the copolymer and pGL4 plasmid were characterized by dynamic light scattering and ζ-potential measurements. The polyplexes retained the thermal phase transition behavior conferred by the copolymer; however, they exhibited a two-step transition process not seen with the copolymer. The polyplexes also showed appreciable transfection efficiency with low cytotoxicity.

Cytoprotection against beta-amyloid (Aβ) peptide-mediated oxidative damage and autophagy by Keap1 RNAi in human glioma U87mg cells.

Extensive oxidative stress has been considered a primary pathological factor for many neurodegenerative disorders (NDDs). We speculated that the oxidative damage to brain cells can be managed by promoting the endogenous cellular antioxidants through the RNA interference (RNAi) against Keap1 (kelch-like ECH-associated protein). Keap1 acts as a negative regulator of Nrf2 (NF-E2-related factor 2) that represses the activation of the antioxidant responsive element (ARE). Here, we investigated whether Keap1 knockdown enhances the cellular antioxidant capacity and provides the neuroprotection against oxidative stress from hydrogen peroxide and beta-amyloid (Aβ) peptide in U87mg cells. We found that the Keap1 siRNA pre-treated group displayed higher expression of diverse antioxidant genes and an increased antioxidant capacity compared to the control group. Moreover, the Keap1 RNAi exerted a cytoprotective effect against H2O2 treatment. In Aβ peptide treatment experiments, the Keap1 siRNA pre-treated groups maintained acceptable cell viability, relatively intact cellular morphology, and controlled oxidative damage levels while the control groups suffered from Aβ peptide-mediated neurotoxicity. Keap1 RNAi also attenuated the oxidative stress-mediated autophagy as well. These findings suggest that Keap1 RNAi can serve as a therapeutic strategy for relieving oxidative stress-associated symptoms in many NDDs.