A mucoadhesive patch loaded with freeze-dried liposomes for the local treatment of oral tumors.

Oral cancers affect millions of people globally, with increasing incidences among adults aged 35 and above. Poor drug uptake by lesions in the oral cavity following systemic administration, as well as limited localized treatment modalities for oral tumors, result in poor patient quality of life and high mortality. Here, we describe a solid, dissolvable, bioadhesive alginate patch containing freeze-dried doxorubicin-loaded liposomes as a local treatment for oral tumors located on the tongue. By varying the alginate-to-liposome ratio in the mucoadhesive patch, we could control the degree of bioadhesion to the tongue and the release profile of the drug-loaded liposomes from the matrix. In vitro, exposing squamous cell carcinoma (SCC) to the alginate mucoadhesive patch or tablet resulted in dose-dependent cancer-cell death. In vivo, the efficacy of the local treatment was demonstrated in mice bearing orthotopic SCC tumors in the tongue. The bioadhesive patch, applied directly above the lesion, significantly reduced the tumor size and treatment-associated side effects compared to implanted patches or systemic drug administration. This study demonstrates that local bioadhesive therapies are effective in treating cancers of the oral cavity.

Lung targeted liposomes for treating ARDS.

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1ß cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.

Foliar Delivery of siRNA Particles for Treating Viral Infections in Agricultural Grapevines.

Grapevine leafroll disease (GLD) is a globally spreading viral infection that causes major economic losses by reducing crop yield, plant longevity and berry quality, with no effective treatment. Grapevine leafroll associated virus-3 (GLRaV-3) is the most severe and prevalent GLD strain. Here, we evaluated the ability of RNA interference (RNAi), a non-GMO gene-silencing pathway, to treat GLRaV-3 in infected Cabernet Sauvignon grapevines. We synthesized lipid-modified polyethylenimine (lmPEI) as a carrier for long double-stranded RNA (dsRNA, 250-bp-long) that targets RNA polymerase and coat protein genes that are conserved in the GLRaV-3 genome. Self-assembled dsRNA-lmPEI particles, 220 nm in diameter, displayed inner ordered domains spaced 7.3±2 nm from one another, correlating to lmPEI wrapping spirally around the dsRNA. The particles effectively protected RNA from degradation by ribonucleases, and Europium-loaded particles applied to grapevine leaves were detected as far as 60-cm from the foliar application point. In three field experiments, a single dose of foliar administration knocked down GLRaV-3 titer, and multiple doses of the treatment kept the viral titer at baseline and triggered recovery of the vine and berries. This study demonstrates RNAi as a promising platform for treating viral diseases in agriculture.

Mucoadhesive Hybrid Polymer/Liposome Pastes Based on Modified Polysaccharides.

Mucoadhesive hybrid polymer/liposome paste is a new drug delivery system presenting controllable and tailorable delivery mechanism. By using mucoadhesive material, the delivery can be more specific and local. Here, we present a study investigating the effect of polymer type, concentration, functional end group, and cross-linking on the release profile of nanoliposomes from polymer pastes. Polymer pastes can be expected to combine the mucoadhesion mechanisms of dry and wet dosage forms but have not been studied extensively. To better understand the mucoadhesion of pastes, we investigated a series of pastes based on the same polymer and used different chemical modifications that can produce interactions at different levels. Native and thiolated polymers presented enhanced mucoadhesion in a wet environment in comparison to acrylated polymers which dissolved rapidly because of the enhanced solubility of PEG chains in water. Paste cross-linking resulted in a sustained release profile compared to non-cross-linked pastes. Pectin-SH pastes, especially 3% (w/v), showed a linear liposomal release profile which is ascribed to the combination of ionic cross-linking and disulfide bridging. By configuring the polymer type or concentration, we can control the release mechanisms and achieve distinct inherent properties which can be applied for diverse medical applications.

Mucoadhesive alginate pastes with embedded liposomes for local oral drug delivery.

Oral cancers are extremely common among adults with increasing incidences due to human papillomavirus, while treatment modalities are limited. This study aims to develop a new oral mucoadhesive delivery system based on the combination of alginate and liposomes. The polymer provides adhesion properties and induces local release of the drug-loaded carriers, while the liposomes protect the drug from degradation and improve its absorption into the cells. Three hybrid alginate/liposomes delivery systems were investigated: a hybrid paste, which presented excellent adhesive capabilities, yet fast burst release of 90% after 2h; a hybrid hydrogel, demonstrating controllable release rates of 5%, 30% or 60% after 2h but poor mucoadhesive properties. These findings led to the development of a hybrid cross-linked paste. Polymer retention studies demonstrated that 80% of the crosslinked paste was retained on tongue tissue compared to 50% retention of the non-cross-linked pastes, verifying its superior mucoadhesion. The hybrid cross-linked paste presented controllable release rate of 20% after 2h. Alginate paste incorporating doxorubicin loaded liposomes presented similar release rates and were highly effective in promoting cancer cell death. Thus, our innovative formulation, including both desired characteristics of mucoadhesion and sustained liposomes release, is an important milestone in the development of a new potential treatment for oral cancer.

Directed evolution of nitrobenzene dioxygenase for the synthesis of the antioxidant hydroxytyrosol.

Nitrobenzene dioxygenase (NBDO) is known to add both atoms of molecular oxygen to the aromatic ring of nitrobenzene to form catechol. It is assembled by four subunits of which the alpha subunit is responsible for catalysis. As an oxidizing enzyme, it has a potential use in the detoxification of industrial waste and the synthesis of pharmaceuticals and food ingredients; however, not much work has been done studying its structure-function correlations. We used several protein engineering approaches (neutral drift libraries, random libraries, two types of focused libraries, and family shuffling) to engineer NBDO for the production of the highly potent antioxidant, hydroxytyrosol (HTyr), from the substrate 3-nitrophenethyl alcohol (3NPA). We obtained a triple mutant, F222C/F251L/G253D, which is able to oxidize 3NPA 375-fold better than wild type with a very high regioselectivity. In total, we identified four positions which are important for acquisition of new specificities, of which only one is well-known and studied. Based on homology modeling, it is suggested that these mutations increase activity by vacating extra space within the active site for the larger substrate and also by hydrogen bonding to the substrate. The best variant had acquired a stabilizing mutation which was beneficial only in this mutant. Thus, we have achieved two goals, the first is the enzymatic production of HTyr, and the second is valuable information regarding the structure-function correlations of NBDO.

Protein engineering of nirobenzene dioxygenase for enantioselective synthesis of chiral sulfoxides.

Nitrobenzene dioxygenase (NBDO) from Comamonas sp. is shown here to perform enantioselective oxidation of aromatic sulfides. Several para-substituted alkyl aryl sulfides were examined and it was found that the activity of the enzyme is dependent on the size of the substrate. Saturation mutagenesis was performed on different residues in the active site in order to improve activity and selectivity. Mutagenesis at position 258 in the α-hydroxylase subunit of NBDO improved both activity and enantioselectivity. Substitutions in position 293 improved the activity on all substrates and had diverse influence on enantioselectivity. Mutagenesis in position 207 provided two interesting variants, V207I and V207A, with opposite enantioselectivities. Furthermore, combining two favorable mutations, N258A and F293H, provided an improved variant with both higher activity (5.20 ± 0.01, 2.12 ± 0.21, 2.64 ± 0.14 and 4.01 ± 0.34 nmol min(-1) mg protein(-1) on thioanisole, ptolyl, Cl-thioanisole and Br-thioanisole, respectively, which is 1.7, 4.6, 7.1 and 26.7-fold compared with wild type) and improved enantioselectivity (e.g. 67% enantiomeric excess for Cl-thioanisole vs. 5% for wild type). Molecular docking and active site volume calculations were used to correlate between the structure of the substrates and the function of the enzymes. The results from this work suggest that the location of pro-chiral sulfides in the active site is coordinated by hydrophobic interactions and by steric considerations, which in turn influences the activity and enantioselectivity of NBDO.

Rapid methods for high-throughput detection of sulfoxides.

Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.

Protein engineering of toluene monooxygenases for synthesis of chiral sulfoxides.

Enantiopure sulfoxides are valuable asymmetric starting materials and are important chiral auxiliaries in organic synthesis. Toluene monooxygenases (TMOs) have been shown previously to catalyze regioselective hydroxylation of substituted benzenes and phenols. Here we show that TMOs are also capable of performing enantioselective oxidation reactions of aromatic sulfides. Mutagenesis of position V106 in the alpha-hydroxylase subunit of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 and the analogous position I100 in toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 improved both rate and enantioselectivity. Variant TomA3 V106M of TOM oxidized methyl phenyl sulfide to the corresponding sulfoxide at a rate of 3.0 nmol/min/mg protein compared with 1.6 for the wild-type enzyme, and the enantiomeric excess (pro-S) increased from 51% for the wild type to 88% for this mutant. Similarly, T4MO variant TmoA I100G increased the wild-type oxidation rate by 1.7-fold, and the enantiomeric excess rose from 86% to 98% (pro-S). Both wild-type enzymes showed lower activity with methyl para-tolyl sulfide as a substrate, but the improvement in the activity and enantioselectivity of the mutants was more dramatic. For example, T4MO variant TmoA I100G oxidized methyl para-tolyl sulfide 11 times faster than the wild type did and changed the selectivity from 41% pro-R to 77% pro-S. A correlation between regioselectivity and enantioselectivity was shown for TMOs studied in this work. Using in silico homology modeling, it is shown that residue I100 in T4MO aids in steering the substrate into the active site at the end of the long entrance channel. It is further hypothesized that the main function of V106 in TOM is the proper positioning or docking of the substrate with respect to the diiron atoms. The results from this work suggest that when the substrate is not aligned correctly in the active site, the oxidation rate is decreased and enantioselectivity is impaired, resulting in products with both chiral configurations.