Bifunctional small molecules that mediate the degradation of extracellular proteins.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

Dinitrophenol-Hyaluronan Conjugates as Multivalent Antibody-Recruiting Glycopolymers for Targeted Cancer Immunotherapy.

Multivalent antibody-recruiting glycopolymers (MARGs) composed of hyaluronic acid (HA) grafted with multiple copies of dinitrophenol (DNP) were developed for targeted cancer immunotherapy. Structure-activity studies demonstrated that the MARGs were able to specifically recognize CD44-positive cancer cells and displayed remarkable antibody-recruiting capacities and tumor cell killing activities dependent on the introduced multivalent effect and the length of PEG linker. One of the MARGs, HA-[PEG3 -DNP]8 , showed the best capacity for clustering anti-DNP antibodies onto CD44-positive cancer cells and displayed potent in vitro anti-cancer activity by triggering complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, we found that HA-[PEG3 -DNP]8 significantly inhibited the xenograft tumor growth of Babl/c nude mice bearing triple negative breast cancer cells, while it did not cause detectable histological cytotoxicity. Given the easy access of this type of natural glycopolymer and the practical synthesis approach, these MARGs provide promising immunotherapeutics for cancer immunotherapy.

Heavy Metals and 2, 4-Dinitrophenol Impact on Some Physiological and Biochemical Parameters in Capsicum Species

Abstract The accumulation of heavy metals and of pesticides in the soil have a negative impact on Capsicum chinense var. Aji Mochero and Capsicum baccatum var. pendulum Bishop's Crown species, the present work highlighting the exerted influence on the superoxid-dismutase activity, catalase and peroxidase - as enzymes implied in the removal of H2O2, of O2- radical and of ion HO- that have devastating effects on the vegetable cell, on the Krebs cycle's dehydrogenases - as main way to produce energy, respectively of the chlorophyll a, b and carotenes - as photoassimilatory pigments, but also as biologic antioxidants.

Molecular mechanistic insights into uncoupling of ion transport from ATP synthesis.

A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F1FO-ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications.

In vitro detection of allergen sensitized basophils by HSA-DNP antigen-anchored liquid crystal microdroplets.

A simple and easy to handle biosensing technique for in vitro detection of HSA-DNP antigen induced allergen reactions in patients has been developed through the detection of sensitized basophils expressed with anti-IgE receptor (FcεRI) by using human serum albumin-dinitrophenol (HSA-DNP) antigen-anchored liquid crystal (LC) microdroplets emulsion. The radial to bipolar transition in nematic 4-cyano-4'-pentyl biphenyl liquid crystal molecules (5CB) confined in HSA-DNP antigen anchored LC microdroplets (8.5 pg HSA-DNP/LC microdroplet) is found to be sensitive in PBS solution in detection of allergen sensitized basophils expressed with a minimum amount of anti HSA-DNP (anti-IgE) receptor (≥4.5 pg/basophil). The detection of allergen sensitized basophils was possible within a contact time of 30 min in presence of control cells and with 10% solution of human blood plasma. The HSA-DNP antigen anchored LC microdroplets in presence of macrophages or non-sensitized basophils did not show radial to bipolar transition in 5CB molecules in PBS or solution with 10 wt% human blood plasma. Thus HSA-DNP antigen anchored LC microdroplets biosensor may be used for in vivo detection of stage I allergen reaction basophils in blood samples.

Making Weak Antigens Strong: Modifying Antigens by Dinitrophenol or Arsynyl Coupling.

Many compounds on their own do not have all of the properties needed to induce a strong antibody response. However, small changes in the structure of an antigen can often greatly alter the immunogenicity of a compound. Common methods for doing so include the addition of small modifying groups such as dinitrophenol or arsenate to the molecules. These techniques either alter regions of the immunogen to provide better sites for T-cell binding or expose new epitopes for B-cell binding. The techniques are rapid and easy, and have been used extensively as a general procedure to increase the chances of raising antisera, particularly against well-conserved antigens.

In Silico Design, Synthesis and Bioactivity of N-(2, 4-Dinitrophenyl)-3-oxo- 3-phenyl-N-(aryl) Phenyl Propanamide Derivatives as Breast Cancer Inhibitors.

BACKGROUND: Breast cancer is a systemic disease which has challenged physicians worldwide as it is the most predominant cancer in women often leading to fatality. One of the types of treatment is chemotherapy which includes targeted oral or intravenous cancer-killing drugs. Treatment options are often limited to surgery and/or chemotherapy. OBJECTIVE: The discovery and design of new small molecule estrogen inhibitors is necessitated in order to circumvent the problem of drug-induced resistance in chemotherapy resulting in disease relapse. Chemoinformatics facilitates the design, selection and synthesis of new drug candidates for breast cancer by providing efficient in silico techniques for prediction of favourable ADMET properties, and structural descriptors to profile druggability of a compound. METHOD: Several molecules selected from docking studies were synthesized and evaluated for their biological activities on the MCF-7 (human breast cancer) cell line. RESULTS: These estrogen inhibitors displayed good inhibitory activity with high selectivity and hence can be further progressed as drug candidates effective against breast cancer. CONCLUSION: It is for the first time that N-(2, 4-dinitrophenyl)-3-oxo-3-phenyl-N-(aryl) phenylpropanamide derivatives were reported to be biological active as potential breast cancer inhibitors.

Suppression of gyrase-mediated resistance by C7 aryl fluoroquinolones.

Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance.

Non-Faradaic Electrochemical Detection of Exocytosis from Mast and Chromaffin Cells Using Floating-Gate MOS Transistors.

We present non-faradaic electrochemical recordings of exocytosis from populations of mast and chromaffin cells using chemoreceptive neuron MOS (CνMOS) transistors. In comparison to previous cell-FET-biosensors, the CνMOS features control (CG), sensing (SG) and floating gates (FG), allows the quiescent point to be independently controlled, is CMOS compatible and physically isolates the transistor channel from the electrolyte for stable long-term recordings. We measured exocytosis from RBL-2H3 mast cells sensitized by IgE (bound to high-affinity surface receptors FcεRI) and stimulated using the antigen DNP-BSA. Quasi-static I-V measurements reflected a slow shift in surface potential () which was dependent on extracellular calcium ([Ca]o) and buffer strength, which suggests sensitivity to protons released during exocytosis. Fluorescent imaging of dextran-labeled vesicle release showed evidence of a similar time course, while un-sensitized cells showed no response to stimulation. Transient recordings revealed fluctuations with a rapid rise and slow decay. Chromaffin cells stimulated with high KCl showed both slow shifts and extracellular action potentials exhibiting biphasic and inverted capacitive waveforms, indicative of varying ion-channel distributions across the cell-transistor junction. Our approach presents a facile method to simultaneously monitor exocytosis and ion channel activity with high temporal sensitivity without the need for redox chemistry.

Fluorimetric screening assay for protein carbonyl evaluation in biological samples.

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.

D-amino carboxamide-based recruitment of dinitrophenol antibodies to bacterial surfaces via peptidoglycan remodeling.

During the past few decades there has been a rapid emergence of multidrug resistant bacteria afflicting human patients. At the same time, reduced output from pharmaceutical industry in this area precipitated a sharp decrease in the approval of new antibiotics. The combination of these factors potentially compromises the ability to effectively combat bacterial infections. While traditional drug discovery efforts continue in the pursuit of small molecule agents that disrupt bacterial growth, non-traditional efforts could serve to complement antimicrobial strategies. We recently demonstrated our ability to remodel the surface of bacterial cells using unnatural D-amino acids displaying the antigenic dinitrophenyl (DNP) handle. These immune stimulant D-amino acids derivatives were metabolically incorporated onto the peptidoglycan of bacteria via a promiscuous surface-anchored transpeptidase. The covalent modification of DNP moieties onto the peptidoglycan led to the anti-DNP antibody opsonization of the bacterial cell surface. Herein, we show that the amidation of the C-terminus to generate DNP-displaying D-amino carboxamide drastically improves antibody recruitment. Antibody opsonization using the D-amino carboxamide agent is observed at lower concentrations than the D-amino acid counterpart. In addition, the recruitment of endogenous antibodies in pooled human serum to the DNP-modified bacterial cell surface is demonstrated for the first time. We envision that the C-terminus amidation of DNP-conjugated D-amino acids could potentially facilitate translation of these results to in vivo animal disease models.

Glutamine suppresses dinitrophenol fluorobenzene-induced allergic contact dermatitis and itching: inhibition of contact dermatitis by glutamine.

BACKGROUND: Cytoplasmic phospholipase A(2) (cPLA(2)) is importantly implicated in a variety of inflammatory diseases by liberating arachidonic acid from phospholipids. The increased cPLA(2) activities as well as increased levels of cPLA(2) metabolites are associated with pathogenesis of many inflammatory skin disorders including atopic dermatitis. The non-essential amino acid l-glutamine (Gln) has been reported to have an anti-inflammatory activity. Regarding the molecular mechanism of Gln, we have recently shown that Gln effectively inhibits cPLA(2) phosphorylation and activity. OBJECTIVE: To examine whether Gln could suppress allergic contact dermatitis (CD) induced on mouse ears by dinitrophenol fluorobenzene (DNFB). METHODS: Mice were sensitized five times on their ears with a 0.15% solution of DNFB in a 3 day interval. To examine Gln effects, Gln solution (4% in saline) was applied three times a day onto both sides of DNFB-applied ears from the last day of DNFB application. The inflammatory reactions of ears were evaluated by measuring ear thickness and hematoxylin and eosin (H&E) staining. Mouse scratching behavior was objectively evaluated using a MicroAct apparatus. cPLA(2) phosphorylation and activity were analyzed using Western blotting and a cPLA(2) assay kit, respectively. RESULTS: Topical application of Gln significantly attenuated inflammatory symptoms (ear thickness, histological inflammatory skin reactions) as well as itching. Gln inhibited cPLA(2) phosphorylation and enzymatic activity. Arachidonyl trifluoromethyl ketone (AACOCF(3)) inhibited cPLA(2) activity in DNFB-challenged ears and attenuated DNFB-induced ear inflammation and itching. CONCLUSION: The results indicate that Gln suppresses DNFB-induced dermatitis and itching, at least in part, by inhibiting cPLA(2) activity.

Theoretical studies on 2-diazo-4,6-dinitrophenol derivatives aimed at finding superior propellants.

In an attempt to find superior propellants, 2-diazo-4,6-dinitrophenol (DDNP) and its -NO(2), -NH(2), -CN, -NC, -ONO(2), and -NF(2) derivatives were studied at the B3LYP/6-311++G level of density functional theory (DFT). Sensitivity was evaluated using bond dissociation enthalpies (BDEs) and molecular surface electrostatic potentials. The C-NO(2) bond appears to be the trigger bond during the thermolysis process for these compounds, except for the -ONO(2) and -NF(2) derivatives. Electrostatic potential results show that electron-withdrawing substituents make the charge imbalance more anomalous, which may change the strength of the bond, especially the weakest trigger bond. Most of the DDNP derivatives have the impact sensitivities that are higher than that of DDNP, making them favorable for use as solid propellants in micro-rockets. The theoretical densities (ρ), heats of formation (HOFs), detonation energies (Q), detonation pressures (P), and detonation velocities (D) of the compounds were estimated. The effects of various substituent groups on ρ, HOF, Q, D, and P were investigated. Some derivatives exhibit perfect detonation properties. The calculated relative specific impulses (I (r,sp)) of all compounds except for -NH(2) derivatives were higher than that of DDNP, and also meet the requirements of propellants.

A high throughput screen for biomining cellulase activity from metagenomic libraries.

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

Synthesis and characterization of spherical 2-diazo-4,6-dinitrophenol (DDNP).

Spherical 2-diazo-4,6-dinitrophenol (DDNP) with good flowability and controlled bulk density (0.65-0.95 g/cm(3)) has been prepared at factory scale by the modified method using 4-methylphenol as crystal control ingredient. Results showed that the yield of product was increased by 5-10%, and the waste water was significantly decreased due to circulation use of waste water compared with traditional method. Synthesized spherical DDNP was characterized by IR, laser granularity measurement, SEM, HPLC and XRD. IR spectrum confirmed the structural features of spherical DDNP. The particle analysis revealed that the modified method could offer spherical DDNP with average particle size of 350 microm and high purity (>98.52%). The XRD peaks of spherical DDNP have similar diffraction angles as those of traditional DDNP. The DSC profile of spherical DDNP showed the exothermic decomposition in the temperature range of 161.2-188.5 degrees C. The product can be pressed at over 40MPa without dead pressed phenomenon, and the minimum detonating charge of spherical DDNP was measured to be about 0.15g. Furthermore, impact sensitivity test suggested that spherical DDNP is less sensitive than traditional DDNP.

Assessment of siRNA pharmacokinetics using ELISA-based quantification.

Here, we developed a novel ELISA-based assay for quantifying double-stranded intact siRNAs for in vivo pharmacokinetic analysis. The assay makes use of dual-labeled unmethylated or methylated siRNA, 5'-end-labeled on one strand with biotin (capture marker), and with dinitrophenol (detection marker), on the other end. This ELISA-based assay was linear over the range of 10-100 fmol/ml, with a sensitivity (5.4 fmol/ml) 629-fold higher than fluorometric quantification methods. The coefficient of variation (CV) of the ELISA quantification was 9.4% for intra-assay and 12.1% for inter-assay. The assay was specific for double-stranded siRNAs. The intensity of the detected signal was reduced to background levels in the presence of single-stranded RNA. The ELISA-based assay revealed that the levels of methylated forms of siRNAs after transfection into A549 and HeLa cells were significantly higher than those of unmethylated siRNA forms. Applying this assay to a study of the pharmacokinetic profiles of intravenously administered siRNAs, we found that the higher blood concentrations were achieved using the methylated form of siRNAs than unmethylated form. Moreover, methylated siRNAs complexed to DOTAP-based cationic liposomes showed significantly higher and prolonged blood concentration-time profile, with 2.2-fold lower clearance rate (0.11+/-0.02 ml/min) as compared to the uncomplexed form. These results demonstrate the utility of an ELISA-based assay for evaluating chemically modified siRNAs and cationic delivery systems, particularly from a pharmacokinetic perspective.

Nitro derivatives from the Arctic ice bacterium Salegentibacter sp. isolate T436.

Twenty-five aromatic nitro, dinitro and trinitro compounds were isolated in low yields of less than 1 mg l(-1) from a Salegentibacter sp. strain T436 derived from Arctic pack ice. Their structures were elucidated by MS and NMR techniques. Seven of these compounds, namely, 2-hydroxy-3-(4'-hydroxy-3'-nitrophenyl)-propionic acid methyl ester (6), 2-chloro-3- (4'-hydroxy-3'-nitrophenyl)propionic acid methyl ester (7), 3-(4'-hydroxy-3',5'-dinitrophenyl)-propionic acid methyl ester (14), 4'-hydroxy-3',5'-dinitrophenylethylchloride (16), (4'-hydroxy-3',5'-dinitrophenyl)-2-chloropropionic acid methyl ester (17), N-acetyl-3',5'-dinitrotyramine (18) and 2,6-dinitro-4-(2'-nitroethenyl)phenol (19) are new, and five are reported in this study from a natural source for the first time.

Folate-targeted hapten immunotherapy of adjuvant-induced arthritis: comparison of hapten potencies.

We have previously reported that disease symptoms can be greatly ameliorated in rodents with adjuvant-induced arthritis (AIA) by first immunizing the rodents against fluorescein and then treating the animals with folate-fluorescein. In this targeted hapten therapy, folate-fluorescein was shown to decorate folate receptor (FR)-expressing activated macrophages with fluorescein (an immunogenic hapten), leading to binding of antifluorescein antibodies and the consequent elimination of the activated macrophages by Fc receptor-expressing immune cells. In the current study, we compare the therapeutic potencies of a variety of FR-targeted haptens in treating the symptoms of AIA in rats. Rats were immunized with either dinitrophenyl (DNP) or trinitrophenyl (TNP) conjugated to keyhole limpet hemocyanin followed by induction of AIA with heat-inactivated Mycobacterium butyricum. Following development of arthritis, rats were treated with one of five folate-hapten conjugates (folate-DNP1, folate-DNP2, folate-DNP3, folate-FITC, or folate-TNP) at two different doses (30 nmol/kg or 200 nmol/kg) 5x/week for 25 days. Symptoms of AIA in treated rats, including paw swelling, arthritis score, splenomegaly, bone erosion, and FR(+) activated macrophage density in inflamed tissues, were quantitated over the course of therapy. Although all folate-hapten conjugates promoted a reduction in disease symptoms, folate-TNP and folate-FITC proved to be more potent than any of the 3 folate-DNP conjugates. We conclude that both folate-TNP and folate-FITC constitute promising haptens for use in FR-targeted immunotherapy of arthritis.

Fenton degradation of 4,6-dinitro-o-cresol with Fe(2+)-substituted ion-exchange resin.

The Fenton degradation of 4,6-dinitro-o-cresol (DNOC) was studied under different experimental conditions using Amberlyst 15 ion-exchange resin containing ferrous ion. DNOC was found to be effectively degraded under most conditions, and it was observed that, with the addition of HCl, the desorption of ferrous ion from the resin into the solution played a major role in this degradation. The total iron concentration in the reaction solution was found to increase with the addition of HCl, and a pseudo-first-order kinetic model was applied to the desorption of ferrous ion from the resin on the basis of the assumption of a first-order ion-exchange process. The degradation rate of DNOC also increased as a function of HCl. A kinetic model was developed to simulate the degradation of DNOC under different operating conditions, assuming the first-order desorption of ferrous ion. Different cations were compared with H(+), and H(+) was found to be the most efficient at facilitating the degradation reaction at low concentrations, whereas Ca(2+) was found to be most efficient at high concentrations. pH was measured during the reaction, and its effect on degradation was explored. It was found that a lower pH could lead to faster degradation of the target compound. Degradation of DNOC under different delivery rates of H(2)O(2) was studied, and optimal conditions were determined. The results also showed that the delivery rate of H(2)O(2) did not affect the ion-exchange process of the resin.

Extracellular barriers to in Vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver.

Polyplex-mediated gene therapy is a promising alternative to viral gene therapy. One challenge to these synthetic carriers is reduced transfection efficiencies in vivo compared to those achieved in vitro. Many of the intracellular barriers to gene delivery have been elucidated, but similar quantification of extracellular barriers to gene delivery remains a need. In this study, the unpackaging of polyplexes by serum proteins, soluble glycosaminoglycans, and an extracellular matrix extract was demonstrated by a YOYO-1 fluorescence quenching assay. Additionally, exposing polyplexes to serum or proteoglycans before in vitro transfection caused decreased cellular uptake of DNA. Lastly, PEI polyplexes and PEGylated PEI polyplexes were injected into the portal vein of mice, and the intrahepatic distributions of labeled DNA and polymer were assessed by confocal microscopy. PEI polyplexes delivered DNA to the liver, but extensive vector unpackaging was observed, with PEI primarily colocalized with the extracellular matrix. PEGylated polyplexes mediated less DNA delivery to the liver, possibly due to premature vector unpackaging in the blood. Through this work, both the blood and the extracellular matrix have been determined to be significant extracellular barriers to polyplex-mediated in vivo gene delivery to the liver.