Synthesis and hybridizing properties of P-stereodefined chimeric [PS]-{DNA:RNA} and [PS]-{DNA:(2'-OMe)-RNA} oligomers.

Oxathiaphospholane derivatives of 2'-OMe-ribonucleosides and 2'-O-TBDMS-ribonucleosides (MN-OTP and TN-OTP, respectively; nucleobase protected) were synthesized and separated into pure P-diastereomers. X-ray analysis showed the R P absolute configuration of the phosphorus atom in the fast-eluting diastereomer of TA-OTP. The fast- and slow-eluting P-diastereomers of MN-OTP and TN-OTP were used in the solid-phase synthesis of phosphorothioate dinucleotides (MNPST and NPST, respectively), which were subsequently hydrolyzed with R P-selective phosphodiesterase svPDE and S P-selective nuclease P1 to determine the absolute configuration of the phosphorus atoms. P-Stereodefined phosphorothioate ([PS]) 10-mer chimeric oligomers [PS]-{DNA:(2'-OMe)-RNA} and isosequential [PS]-{DNA:RNA} containing two MNPS or NPS units were synthesized. Melting experiments performed for their complexes with Watson-Crick paired DNA matrix showed that MNPS or NPS units decrease the thermal stability of the duplexes (ΔT m = -0.5 ÷ -5.5 °C per modification) regardless of the absolute configuration of the P-atoms. When the (2'-OMe)-RNA matrix was used an increase in T m was noted in all cases (ΔT m = +1 ÷ +7 °C per modification). The changes in thermal stability of the duplexes formed by [PS]-chimeras with DNA and (2'-OMe)-RNA matrices do not correlate with the absolute configuration of the phosphorus atoms.

Thiomorpholino oligonucleotides as a robust class of next generation platforms for alternate mRNA splicing.

Recent advances in drug development have seen numerous successful clinical translations using synthetic antisense oligonucleotides (ASOs). However, major obstacles, such as challenging large-scale production, toxicity, localization of oligonucleotides in specific cellular compartments or tissues, and the high cost of treatment, need to be addressed. Thiomorpholino oligonucleotides (TMOs) are a recently developed novel nucleic acid analog that may potentially address these issues. TMOs are composed of a morpholino nucleoside joined by thiophosphoramidate internucleotide linkages. Unlike phosphorodiamidate morpholino oligomers (PMOs) that are currently used in various splice-switching ASO drugs, TMOs can be synthesized using solid-phase oligonucleotide synthesis methodologies. In this study, we synthesized various TMOs and evaluated their efficacy to induce exon skipping in a Duchenne muscular dystrophy (DMD) in vitro model using H2K mdx mouse myotubes. Our experiments demonstrated that TMOs can efficiently internalize and induce excellent exon 23 skipping potency compared with a conventional PMO control and other widely used nucleotide analogs, such as 2'-O-methyl and 2'-O-methoxyethyl ASOs. Notably, TMOs performed well at low concentrations (5-20 nM). Therefore, the dosages can be minimized, which may improve the drug safety profile. Based on the present study, we propose that TMOs represent a new, promising class of nucleic acid analogs for future oligonucleotide therapeutic development.

Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention.

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.

Synthesis and Characterization of Thiophosphoramidate Morpholino Oligonucleotides and Chimeras.

This Article outlines the optimized chemical synthesis and preliminary biochemical characterization of a new oligonucleotide analogue called thiophosphoramidate morpholinos (TMOs). Their rational design hinges upon integrating two well-studied pharmacophores, namely, phosphorothioates (pS) and morpholinos, to create morpholino-pS hybrid oligonucleotides. Our simple synthesis strategy enables the easy incorporation of morpholino-pS moieties and therapeutically relevant sugar modifications in tandem to create novel oligonucleotide (ON) analogues that are hitherto unexplored in the oligotherapeutics arena. Exclusively TMO-modified ONs demonstrate high stability toward 3'-exonuclease. Hybridization studies show that TMO chimeras consisting of alternating TMO and DNA-pS subunits exhibit higher binding affinity toward complementary RNA relative to the canonical DNA/RNA duplex (∼10 °C). Oligonucleotides that consist entirely of TMO linkages also show higher RNA binding affinity but do not recruit ribonuclease H1 (RNase H1). Chimeric TMO analogues demonstrate high gene silencing efficacy, comparable to that of a chimeric 2'-OMe-pS/pO control, during in vitro bioassay screens designed to evaluate their potential as microRNA inhibitors of hsa-miR-15b-5p in HeLa cells.

Drug affinity and targeted delivery: double functionalization of silk spheres for controlled doxorubicin delivery into Her2-positive cancer cells.

BACKGROUND: The optimal drug delivery system should be biocompatible, biodegradable, and allow the sustained release of the drug only after it reaches the target cells. Silk, as a natural polymer, is a great candidate for building drug carriers. Genetically engineered silks offer the possibility of functionalization. Previously, we characterized bioengineered silk spheres that were functionalized with H2.1 peptide that selectively delivered a drug to Her2-positive cancer cells. However, drug leakage from the silk spheres showed the need for improved control. RESULTS: To control the drug loading and release, we designed and produced functional silk (DOXMS2) that contains a DOX peptide with an affinity for doxorubicin. The DOXMS2 spheres showed the decreased release of doxorubicin compared with MS2 particles. Next, the DOXMS2 silk was blended with the H2.1MS1 polymer to improve the control of doxorubicin binding and release into Her2-positive cancer cells. The H2.1MS1:DOXMS2 particles showed the highest doxorubicin-loading capacity and binding per cell, which resulted in the highest cytotoxic effect compared with that of other sphere variants. Since drug release at a pH of 7.4 from the blended H2.1MS1:DOXMS2 particles was significantly lower than from blended spheres without DOXMS2 silk, this indicated that such particles could control the release of the drug into the circulatory system before the carrier reached the tumor site. CONCLUSIONS: This strategy, which is based on the blending of silks, allows for the generation of particles that deliver drugs in a controlled manner.

LNA units present in [R P-PS]-(DNA#LNA) chimeras enhance the thermal stability of parallel duplexes and triplexes formed with (2'-OMe)-RNA strands.

The results of CD measurements indicate that 2-4 LNA units distributed along 12 nt P-stereodefined phosphorothioate [R P-PS]-(DNA#LNA) chimeras impose a C3'-endo conformation on the 2'-deoxyribonucleosides. Under neutral and slightly acidic conditions homopurine [R p-PS]-(DNA#LNA) hybridizes with 9-12 nt Hoogsteen-paired (2'-OMe)-RNA strands to form parallel duplexes, which are thermally more stable than the reported earlier analogous complexes containing LNA-free [R P-PS]-DNA oligomers (ΔT m = 7 °C per LNA unit at pH 5.4). Upon addition of the corresponding Watson-Crick-paired (2'-OMe)-RNA strands, parallel triplexes are formed with further increased thermal stability.

Exploring site-specific activation of bis-N,N'-dialkylaminophosphordiamidites and the synthesis of morpholinophosphoramidate oligonucleotides.

Morpholinos are six-membered rings that may provide higher conformational rigidity when incorporated into an oligonucleotide (ODN) backbone. Phosphorodiamidate morpholinos are chemically modified ODNs containing morpholinos in place of 2'-deoxyribose moieties throughout their backbone and have garnered much interest in recent years due to their ability to function as highly effective steric blockers in exon skipping therapy. To further explore the biophysical and biological properties of ODNs derived from morpholino nucleosides, we have replaced the 2'-deoxyribonucleotides of phosphodiester DNA with morpholinonucleotides to generate phosphoramidate ODNs. Here, we evaluate the mechanistic pathways observed during the solution-phase synthesis of morpholinonucleoside phosphoramidites, solid-phase synthesis of morpholinonucleotide phosphoramidates of mA, mG, mC and mT (prefix 'm' represents morpholino) and our first attempts directed at the solid-phase synthesis of chimeric DNA-phosphoramidate ODNs, as well as fully modified 22-mer phosphoramidate ODNs.

Homopurine RP-stereodefined phosphorothioate analogs of DNA with hampered Watson-Crick base pairings form Hoogsteen paired parallel duplexes with (2'-OMe)-RNAs.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5'-O-DMT-N6-methyl-deoxyadenosine and 5'-O-DMT-N2,N2-dimethyl-O6-diphenylcarbamoyl-deoxyguanosine (OTP-NY, NY = DMT-m6dA or DMT-m,m2dGDPC) were synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3',5,-phosphorothioates NXPST (NX = m6dA or m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom was established enzymatically. Diastereomerically pure OTP-NY and standard OTP-N (N = DMT-dABz or DMT-dGBz,DPC) were used in the synthesis of chimeric RP-stereodefined phosphorothioate oligomers ((RP-PS)-DN(NX)A) with hampered Watson-Crick base pairings. It was found that the m6dA units slightly reduce the thermodynamic stability of antiparallel duplexes formed with RNA and (2'-OMe)-RNA matrices, whereas m,m2dG units prevent their formation. The m6dA units stabilize (by up to 4.5 °C per modified unit) the parallel duplexes formed by (RP-PS)-DN(NX)A with Hoogsteen-paired (2'-OMe)-RNA templates compared to the analogous reference duplex containing only unmodified nucleobases. In contrast, the m,m2dG units destabilize such duplexes by up to 3 °C per modified unit. Both units prevent the formation of the corresponding parallel triplexes.

Optimization of spider silk sphere formation processing conditions to obtain carriers with controlled characteristics.

Bioengineered spider silk is a focus of research due to its biocompatibility, biodegradability, and excellent mechanical properties. Functionalized silk can be processed into spheres and employed as selective drug delivery vehicles in targeted cancer treatment. Efficient, repeatable and controllable processing conditions are essential to drug carrier development. This study aimed to optimize the processing conditions of silk spheres formation, scale-up, and automation of the silk spheres production process. The automated micromixing system provided substantial amounts of silk spheres under repetitive production conditions. Micromixing resulted in smaller sphere sizes and narrower sphere size distributions than mixing with a pipette. Furthermore, the particle size and size distribution of silk spheres could be tailored by varying mixing process parameters, such as protein concentration, silk and salting out buffer ratio, mixing speed, and the size of the tubes and mixing zone. In addition, the implementation of ultrafiltration techniques provided a fast and efficient concentration of spheres in water. Furthermore, the shear forces introduced by micromixing did not impede the properties of the Her2 binding peptide (H2.1) since the functionalized H2.1MS1:H2.1MS2 silk spheres selectively were internalized by Her2-positive cancer cells. This study indicated that micromixing in combination with ultrafiltration enabled scale-up of the sphere production process under controllable and repeatable conditions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3211-3221, 2018.

Bioengineering the spider silk sequence to modify its affinity for drugs.

BACKGROUND: Silk is a biocompatible and biodegradable material, able to self-assemble into different morphological structures. Silk structures may be used for many biomedical applications, including carriers for drug delivery. The authors designed a new bioengineered spider silk protein, EMS2, and examined its property as a carrier of chemotherapeutics. MATERIALS AND METHODS: To obtain EMS protein, the MS2 silk monomer (that was based on the MaSp2 spidroin of Nephila clavipes) was modified by the addition of a glutamic acid residue. Both bioengineered silks were produced in an Escherichia coli expression system and purified by thermal method. The silk spheres were produced by mixing with potassium phosphate buffer. The physical properties of the particles were characterized using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and zeta potential measurements. The MTT assay was used to examine the cytotoxicity of spheres. The loading and release profiles of drugs were studied spectrophotometrically. RESULTS: The bioengineered silk variant, EMS2, was constructed, produced, and purified. The EMS2 silk retained the self-assembly property and formed spheres. The spheres made of EMS2 and MS2 silks were not cytotoxic and had a similar secondary structure content but differed in morphology and zeta potential values; EMS2 particles were more negatively charged than MS2 particles. Independently of the loading method (pre- or post-loading), the loading of drugs into EMS2 spheres was more efficient than the loading into MS2 spheres. The advantageous loading efficiency and release rate made EMS2 spheres a good choice to deliver neutral etoposide (ETP). Despite the high loading efficiency of positively charged mitoxantrone (MTX) into EMS2 particles, the fast release rate made EMS2 unsuitable for the delivery of this drug. A faster release rate from EMS2 particles compared to MS2 particles was observed for positively charged doxorubicin (DOX). CONCLUSION: By modifying its sequence, silk affinity for drugs can be controlled.

Delivery of chemotherapeutics using spheres made of bioengineered spider silks derived from MaSp1 and MaSp2 proteins.

AIM: Analysis of the properties and chemotherapeutics delivery potential of spheres made of bioengineered spider silks MS1 and MS2. MATERIALS & METHODS: MS1 and MS2 derived from Nephila clavipes dragline silks - MaSp1 and MaSp2, respectively - formed spheres that were compared in terms of physicochemical properties, cytotoxicity and loading/release of chemotherapeutics. RESULTS: MS2 spheres were more dispersed, smaller, of solid core, of higher beta-sheet structure content, and of opposite (negative) charge than MS1 spheres. Preloaded MS2 showed greater applicability for mitoxantrone, while postloaded for etoposide delivery compared with MS1 spheres. However, MS1 spheres were a better choice for doxorubicin delivery than MS2. CONCLUSION: Bioengineered silks can be tailored to develop a system with optimal drug loading and release properties.

P-Stereodefined phosphorothioate analogs of glycol nucleic acids-synthesis and structural properties.

Enantiomerically pure, protected acyclic nucleosides of the GNA type (glycol nucleic acids) (GN'), obtained from (R)-(+)- and (S)-(-)-glycidols and the four canonical DNA nucleobases (Ade, Cyt, Gua and Thy), were transformed into 3'-O-DMT-protected 2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane derivatives (OTP-GN') containing a second stereogenic center at the phosphorus atom. These monomers were chromatographically separated into P-diastereoisomers, which were further used for the synthesis of P-stereodefined "dinucleoside" phosphorothioates GNPST (GN = GA, GC, GG, GT). The absolute configuration at the phosphorus atom for all eight GNPST was established enzymatically and verified chemically, and correlated with chromatographic mobility of the OTP-GN' monomers on silica gel. The GNPS units (derived from (R)-(+)-glycidol) were introduced into self-complementary PS-(DNA/GNA) octamers of preselected, uniform absolute configuration at P-atoms. Thermal dissociation experiments showed that the thermodynamic stability of the duplexes depends on the stereochemistry of the phosphorus centers and relative arrangement of the GN units in the oligonucleotide strands. These results correlate with the changes of conformation assessed from circular dichroism spectra.

Blending two bioengineered spider silks to develop cancer targeting spheres.

Bioengineered spider silk is a material that combines superb mechanical properties, biocompatibility and biodegradability with simple production and purification procedures. Moreover, genetic engineering enables the functionalization of silk by adding the sequence encoding the desired attribute. Functionalized silk can self-assemble into spheres that may serve as a carrier for targeted cancer diagnostics/therapy. MaSp1- and MaSp2-based bioengineered silk proteins (MS1 and MS2, respectively) and their anti-cancer oriented hybrid variants were studied. A new approach was applied that blended the two functionalized silk types (MS1 and MS2) at different weight ratios. We selected spheres formed by the blending of functionalized MS1 and MS2 silks at a ratio of 8 : 2 that bound to the target cells at the same level as functionalized MS1 spheres but had greatly improved properties, including size, size distribution, colloidal stability and production efficiency. Compared with functionalized MS1 spheres, functionalized MS1:MS2 particles efficiently killed targeted cells when loaded with a drug inducing considerably lower non-specific toxicity. This study indicates that the blending of silk materials might be advantageous because it combines the most favorable characteristics of both silks and can lead to the formation of an optimal drug delivery vehicle.

The method of purifying bioengineered spider silk determines the silk sphere properties.

Bioengineered spider silks are a biomaterial with great potential for applications in biomedicine. They are biocompatible,biodegradable and can self-assemble into films, hydrogels, scaffolds, fibers, capsules and spheres. A novel, tag-free, bioengineered spider silk named MS2(9x) was constructed. It is a 9-mer of the consensus motif derived from MaSp2-the spidroin of Nephila clavipes dragline silk. Thermal and acidic extraction methods were used to purify MS2(9x). Both purification protocols gave a similar quantity and quality of soluble silk; however, they differed in the secondary structure and zeta potential value. Spheres made of these purified variants differed with regard to critical features such as particle size, morphology, zeta potential and drug loading. Independent of the purification method, neither variant of the MS2(9x) spheres was cytotoxic, which confirmed that both methods can be used for biomedical applications. However, this study highlights the impact that the applied purification method has on the further biomaterial properties.

Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of LNA-type nucleosides (LNA-OTPs, 2a-d; B' = Thy, Ade(Bz), Cyt(Bz), Gua(dmf), respectively) were synthesized and separated into pure P-diastereomers. X-ray analysis allowed for assignment of the absolute configuration of the phosphorus atom in the detritylated, fast-eluting diastereomer 2a. The diastereomerically pure LNA-OTP monomers were used in solid phase synthesis of P-stereodefined chimeric PS-(DNA/LNA) 11-mers containing 2-3 LNA units. Formally, among the phosphorothioate oligomers the biggest enhancement in thermal stability of Watson-Crick paired duplexes was found for [SP-PS]-(DNA/LNA)/RNA duplexes (on average 8.2 °C per LNA nucleotide), followed by [RP-PS]-(DNA/LNA)/RNA (6.3 °C), [RP-PS]-(DNA/LNA)/DNA (3.8 °C) and [SP-PS]-(DNA/LNA)/DNA (2.4 °C per LNA nucleotide). However, detailed analysis of the thermal dissociation data showed that the thermal stability of (PS-LNA)-containing duplexes does not depend on the spatial orientation of the sulfur atoms. This conclusion received support from CD measurements.

Silk as an innovative biomaterial for cancer therapy.

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.

Polymeric nanoparticles-embedded organogel for roxithromycin delivery to hair follicles.

Drug delivery into hair follicles with the use of nanoparticles (NPs) is gaining more importance as drug-loaded NPs may accumulate in hair follicle openings. The aim was to develop and evaluate a pluronic lecithin organogel (PLO) with roxithromycin (ROX)-loaded NPs for follicular targeting. Polymeric NPs were evaluated in terms of particle shape, size, zeta potential, suspension stability, encapsulation efficiency and in vitro drug release. Lyophilized NPs were incorporated into the PLO and rheological measurements of the nanoparticles-embedded organogels were done. The fate of the NPs in the skin was traced by incorporation of a fluorescent dye into the NPs. As a result, ROX was efficiently incorporated into polymeric NPs characterized by the appropriate size (approximately 300 nm) allowing drug delivery to hair follicles. In ex vivo human skin penetration studies, horizontal skin sections revealed fluorescence deep in the hair follicles. Although the organogel has higher affinity to the lipidic follicular area than an aqueous suspension of NPs, it did not seem to improve penetration of the NPs along the hair shaft. The results proved that it was possible to achieve preferential targeting to the pilosebaceous unit using polymeric NPs formulated either into the aqueous suspension or semisolid topical formulation.

Functional group transformations in derivatives of 6-oxoverdazyl.

Transformations of functional groups, such as OCH2Ph, OCOPh, NO2 and I, in 1,3,5-triphenyl-6-oxoverdazyls 1a-1e were investigated in order to expand the range of synthetic tools for incorporation of the verdazyl system into more complex molecular architectures and to increase spin delocalization. Thus, Pd-catalyzed debenzylation of the OCH2Ph group or basic hydrolysis of the OCOPh group gave the phenol functionality, which was acylated, but could not be alkylated. Orthogonal deprotection of diphenol functionality was also demonstrated in radical 1c. Pt-catalyzed reduction of the NO2 group led to the aniline derivative, which was acylated. Attempted C-C coupling reactions to iodophenyl derivatives 1e and 5e were unsuccessful. Selected verdazyl radicals were characterized by EPR and electronic absorption spectroscopy, and results were analyzed with the aid of DFT computational methods.