A facile and scalable method to synthesize PEGylated PDMAEMA for gene delivery.

In recent years, cationic polymer vectors have been viewed as a promising method for delivering nucleic acids. With the advancement of synthetic polymer chemistry, we can control chemical structures and properties to enhance the efficacy of gene delivery. Herein, a facile, cost-effective, and scalable method was developed to synthesize PEGylated PDMAEMA polymers (PEO-PDMAEMA-PEO), where PEGylation could enable prolonged polyplexes circulation time in the blood stream. Two polymers of different molecular weights were synthesized, and polymer/eGFP polyplexes were prepared and characterized. The correlation between polymers' molecular weight and physicochemical properties (size and zeta potential) of polyplexes was investigated. Lipofectamine 2000, a commercial non-viral transfection reagent, was used as a standard control. PEO-PDMAEMA-PEO with higher molecular weight exhibited slightly better transfection efficiency than Lipofectamine 2000, and the cytotoxicity study proved that it could function as a safe gene vector. We believe that PEO-PDMAEMA-PEO could serve as a model to investigate more potential in the gene delivery area.

Atom transfer radical-polymerized cationic shells on gold nanoparticles for near infrared-triggered photodynamic therapy of tumor-bearing animals.

Gold nanoparticles (AuNPs) were surface-engineered with a cationic corona to enhance the incorporation of photosensitizers for photodynamic therapy (PDT). The cationic corona composed of poly(2-(dimethylamino)ethyl methacrylate) was atom transfer radical-polymerized on the surface of the AuNPs. The cationic corona of the engineered surface was characterized by dynamic light scattering, electron microscopy, Raman spectroscopy, and mass spectroscopy. Chlorin-e6 (Ce6) incorporated onto the surface-engineered AuNPs exhibited higher cell incorporation efficiency than bare AuNPs. Ce6-incorporated AuNPs were confirmed to release singlet oxygen upon NIR irradiation. Compared to Ce6, Ce6-incorporated AuNPs exhibited higher cellular uptake and cytotoxicity against cancer cells in an irradiation time-dependent manner. Near-infrared-irradiated animals administered Ce6-incorporated AuNPs exhibited higher levels of tumor suppression without noticeable body weight loss. This result was attributed to the higher localization of Ce6 at the tumor sites to induce cancer cell apoptosis. Thus, we envision that engineered AuNPs with cationic corona can be tailored to effectively deliver photosensitizers to tumor sites for photodynamic therapy.

Targeting anaplastic lymphoma kinase (ALK) gene alterations in neuroblastoma by using alkylating pyrrole-imidazole polyamides.

Anaplastic lymphoma kinase (ALK) aberration is related to high-risk neuroblastomas and is an important therapeutic target. As acquired resistance to ALK tyrosine kinase inhibitors is inevitable, novel anti-ALK drug development is necessary in order to overcome potential drug resistance against ATP-competitive kinase inhibitors. In this study, to overcome ALK inhibitor resistance, we examined the growth inhibition effects of newly developed ALK-targeting pyrrole-imidazole polyamide CCC-003, which was designed to directly bind and alkylate DNA within the F1174L-mutated ALK gene. CCC-003 suppressed cell proliferation in ALK-mutated neuroblastoma cells. The expression of total and phosphorylated ALK was downregulated by CCC-003 treatment but not by treatment with a mismatch polyamide without any binding motif within the ALK gene region. CCC-003 preferentially bound to the DNA sequence with the F1174L mutation and significantly suppressed tumor progression in a human neuroblastoma xenograft mouse model. Our data suggest that the specific binding of CCC-003 to mutated DNA within the ALK gene exerts its anti-tumor activity through a mode of action that is distinct from those of other ALK inhibitors. In summary, our current study provides evidence for the potential of pyrrole-imidazole polyamide ALK inhibitor CCC-003 for the treatment of neuroblastoma thus offering a possible solution to the problem of tyrosine kinase inhibitor resistance.

Tailoring the Architecture of Cationic Polymer Brush-Modified Carbon Nanotubes for Efficient siRNA Delivery in Cancer Immunotherapy.

The facile and controlled fabrication of homogeneously grafted cationic polymers on carbon nanotubes (CNTs) remains poorly investigated, which further hinders the understanding of interactions between functionalized CNTs with different nucleic acids and the rational design of appropriate gene delivery vehicles. Herein, we describe the controlled grafting of cationic poly(2-dimethylaminoethylmethacrylate) brushes on CNTs via surface-initiated atom transfer radical polymerization integrated with mussel-inspired polydopamine chemistry. The binding of nucleic acids with different brush-CNT hybrids discloses the highly architectural-dependent behavior with dense short brush-coated CNTs displaying the highest binding among all the other hybrids, namely, dense long, sparse long, and sparse short brush-coated CNTs. Additionally, different chemistries of the brush coatings were shown to influence the biocompatibility, cellular uptake, and silencing efficiency in vitro. This platform provides great flexibility for the design of polymer brush-CNT hybrids with precise control over their structure-activity relationship for the rational design of nucleic acid delivery systems.

Targeting the mutant PIK3CA gene by DNA-alkylating pyrrole-imidazole polyamide in cervical cancer.

PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.

A novel amino cellulose derivative using ATRP method: Preparation, characterization, and investigation of its antibacterial activity.

In this study, we prepared a novel amino cellulose derivative (benzyl cellulose-g-poly [2-(N,N-Dimethylamino)ethyl methacrylate]) via a homogeneous ATRP method. The successful synthesis of the novel amino cellulose was confirmed by FT-IR and 1H NMR. This study addressed the different characteristics of the prepared polymer including the thermal stability, solubility, and X-ray diffraction pattern. The antibacterial activity of the synthesized cellulose derivative was investigated using the diffusion disk method against both gram-negative (Escherichia coli, Salmonella enterica) and gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria. Based on the inhibition zone, it was confirmed that the prepared benzyl cellulose-g-PDMAEMA possesses acceptable antibacterial activity against Escherichia coli, Salmonella enterica, and Staphylococcus aureus while Bacillus subtilis is resistant to the prepared polymer. Also according to the inhibition zone, it was shown that benzyl cellulose-g-PDMAEMA has more impact on E. coli and Salmonella enterica than Staphylococcus aureus. Molecular dynamics simulation was also used to study the interaction of the synthesized cellulose derivative with a model membrane which presented atomistic details of the polymer-lipid interactions. According to the results obtained from the molecular dynamics simulation, the polymer was able to destabilize the structure of the membrane and clearly express its signs of degradation.

Chemical Approaches to the Development of Artificial Transcription Factors Based on Pyrrole-Imidazole Polyamides.

To maintain the functions of living organisms, cells have developed complex gene regulatory networks. Transcription factors have a central role in spatiotemporal control of gene expression and this has motivated us to develop artificial transcription factors that mimic their function. We found that three functions could be mimicked by applying our chemical approaches: i) efficient delivery into organelles that contain target DNA, ii) specific DNA binding to the target genomic region, and iii) regulation of gene expression by interaction with other transcription coregulators. We chose pyrrole-imidazole polyamides (PIPs), sequence-selective DNA binding molecules, as DNA binding domains, and have achieved each of the required functions by introducing other functional moieties. The developed artificial transcription factors have potential as chemical tools that can be used to artificially modulate gene expression to enable cell fate control and to correct abnormal gene regulation for therapeutic purposes.

Design, synthesis and anti-cancer activity of pyrrole-imidazole polyamides through target-downregulation of c-kit gene expression.

Pyrrole-imidazole polyamide (PIP) can specifically bind in the B-DNA minor groove that has been used in several biological applications, such as anti-cancer activity, gene expression and translation control, and visualization of complex genomic areas. c-kit is a family member of the Tyrosine Kinase (RTK) type III receptor and plays a vital role in tumor growth, proliferation, differentiation, and cell apoptosis; however, its mutations and overexpression induce tumor dysfunction. Here, we designed and synthesized five matched PIPs that can recognize and bind to the DNA sequence in the oncogene c-kit promoter region, and evaluated their anti-cancer activity. The RTCA assay findings revealed that the PIPs would prevent the proliferation of cancer cells A549 and SGC-7901. EMSA assay showed that the PIPs were actively interacting with the c-kit gene target DNA. RT-PCR and Western blot assays have an effect on expression levels of the c-kit gene in the presence of PIPs. Flow cytometry and wound-healing assays showed that PIPs 4, 5 would cause apoptosis of cancer cells and inhibit the migration of cells, respectively. Overall findings indicate that PIP 5 has a relatively significant intracellular and extracellular impact on the target, contributing to migration and proliferation reduction, and cancer cell apoptosis. In addition, PIP has a certain ability to evolve into c-kit gene-targeting drugs.

Elastomeric polyamide biomaterials with stereochemically tuneable mechanical properties and shape memory.

Biocompatible polymers are widely used in tissue engineering and biomedical device applications. However, few biomaterials are suitable for use as long-term implants and these examples usually possess limited property scope, can be difficult to process, and are non-responsive to external stimuli. Here, we report a class of easily processable polyamides with stereocontrolled mechanical properties and high-fidelity shape memory behaviour. We synthesise these materials using the efficient nucleophilic thiol-yne reaction between a dipropiolamide and dithiol to yield an α,ß - unsaturated carbonyl moiety along the polymer backbone. By rationally exploiting reaction conditions, the alkene stereochemistry is modulated between 35-82% cis content and the stereochemistry dictates the bulk material properties such as tensile strength, modulus, and glass transition. Further access to materials possessing a broader range of thermal and mechanical properties is accomplished by polymerising a variety of commercially available dithiols with the dipropiolamide monomer.

Comb-Like Amphiphilic Polycarbonates with Different Lengths of Cationic Branches for Enhanced siRNA Delivery.

Owing to the biodegradability and good biocompatibility polycarbonates show the versatile class of applications in biomedical fields. While their poor functional ability seriously limited the development of functional polycarbonates. Herein, a new Br-containing cyclic carbonate (MTC-Br) and a polycarbonate atom transfer radical polymerization (ATRP) macro-initiator (PEG-PMTC-Br) is synthesized. Then, by initiating the side-chain ATRP of 2-(dimethyl amino)ethyl methacrylate (DMAEMA) on PEG-PMTC-Br, a series of comb-like amphiphilic cationic polycarbonates, PEG-b-(PMTC-g-PDMAEMA) (GMDMs), with different lengths of cationic branches are successfully prepared. All these poly(ethylene glycol)-b-(poly((5-methyl-2-oxo-1,3-dioxane-5-yl) methyl 2-bromo-2-methylpropanoate/1,3-dioxane-2-one)-g-poly(2-dimethyl aminoethyl methacrylate) (GMDMs) self-assembled nanoparticles (NPs) (≈180 nm, +40 mV) can well bind siRNA to form GMDM/siRNA NPs. The gene silence efficiency of GMDM/siRNA high to 80%, which is even higher than the commercial transfection reagent lipo2000 (76%). But GMDM/siRNA shows lower cell uptake than lipo2000. So, the high gene silence ability of GMDM/siRNA NPs can be attributed to the strong intracellular siRNA trafficking capacity. Therefore, GMDM NPs are potential siRNA vectors and the successful preparation of comb-like polycarbonates also provides a facile way for diverse side-chain functional polycarbonates, expanding the application of polycarbonates.

Electrochromic Response Capability Enhancement with Pentiptycene-Incorporated Intrinsic Porous Polyamide Films.

Enhancing switching response capability of electrochromic (EC) polymers has been a topical issue. Herein, the H-shaped nonplanar pentiptycene scaffold is successfully introduced into the polyamide film derived from N,N'-bis(4-aminophenyl)-N,N'-di(methoxyphenyl)-1,4-phenylenediamine (TPPA), and the resulting copolymer shows enhanced EC behaviors, including lower oxidation potential and shorter switching response time, as compared to the corresponding TPPA-derived homopolymer.

Targeting Breast Cancer Cells with G4 PAMAM Dendrimers and Valproic Acid Derivative Complexes.

BACKGROUND: Our research group has developed some Valproic Acid (VPA) derivatives employed as anti-proliferative compounds targeting the HDAC8 enzyme. However, some of these compounds are poorly soluble in water. OBJECTIVE: Employed the four generations of Polyamidoamine (G4 PAMAM) dendrimers as drug carriers of these compounds to increase their water solubility for further in vitro evaluation. METHODS: VPA derivatives were subjected to Docking and Molecular Dynamics (MD) simulations to evaluate their affinity on G4 PAMAM. Then, HPLC-UV/VIS, 1H NMR, MALDI-TOF and atomic force microscopy were employed to establish the formation of the drug-G4 PAMAM complexes. RESULTS: The docking results showed that the amide groups of VPA derivatives make polar interactions with G4 PAMAM, whereas MD simulations corroborated the stability of the complexes. HPLC UV/VIS experiments showed an increase in the drug water solubility which was found to be directly proportional to the amount of G4 PAMAM. 1H NMR showed a disappearance of the proton amine group signals, correlating with docking results. MALDI-TOF and atomic force microscopy suggested the drug-G4 PAMAM dendrimer complexes formation. DISCUSSION: In vitro studies showed that G4 PAMAM has toxicity in the micromolar concentration in MDAMB- 231, MCF7, and 3T3-L1 cell lines. VPA CF-G4 PAMAM dendrimer complex showed anti-proliferative properties in the micromolar concentration in MCF-7 and 3T3-L1, and in the milimolar concentration in MDAMB- 231, whereas VPA MF-G4 PAMAM dendrimer complex didn't show effects on the three cell lines employed. CONCLUSION: These results demonstrate that G4 PAMAM dendrimers are capableof transporting poorly watersoluble aryl-VPA derivate compounds to increase its cytotoxic activity against neoplastic cell lines.

Seed's morphology-induced core-shell composite particles by seeded emulsion polymerization for drug delivery.

Cross-liked poly(2-hydroxyethyl methacrylate-co-methacrylic acid) seeds with different morphologies such as cauliflower-like, lobed spherical, and spherical were used in seeded emulsion polymerization (SEP) of 2-(dimethylamino)ethyl methacrylate (DMAEMA), 2-hydroxyethyl methacrylate (HEMA), and methacrylic acid (MAA). The morphological structure of produced composite particles was observed using field emission scanning electron microscopy (FE-SEM). The origin of the formation of different morphologies was discussed using various thermodynamic parameters such as solubility parameters and intermolecular forces between polymeric components involved. Also, the effect of the morphology of seed particles on the resultant structures was investigated. Results showed that morphology of fabricated composite particles is induced from morphology of seed particles with larger sizes. Finally, the fabricated composite particles were utilized in the controlled release of DOX. The effect of morphological changes of synthesized composite particles on the cumulative release behavior at acidic environment indicated the pH-sensitive nature of drug release through carriers. The particles with PDMAEMA shell showed the highest release of DOX at pH = 7.4 whereas PMAA shells displayed the least cumulative release. Inversely, the lowest cumulative release at pH = 1.2 was shown by PDMAEMA-coated carriers. Moreover, particles with spherical morphology had better drug release than cauliflower-like ones originated from smart nature of carriers.

Synthesis and Evaluation of Dendrimers for Autophagy Augmentation and Alleviation of Obstructive Lung Diseases.

Preservation of cellular homeostasis requires constant synthesis of fresh proteins and cellular organelles and efficient degradation or removal of damaged proteins and cellular components. This involves two cellular degradation processes or molecular mechanisms: the ubiquitin-proteasome and autophagy-lysosomal systems. Impairment of these catabolic processes has been linked to pathogenesis of a variety of chronic obstructive lung diseases such as COPD (chronic obstructive pulmonary disease) and CF (cystic fibrosis). Proteosomal and autophagic functions (proteostasis) are known to decline with advancing age leading to accumulation of cellular debris and proteins, initiating cellular senescence or death and accelerating lung aging. Obstructive lung diseases associated with airway hyperinflammation and mucus obstruction provide major challenges to the delivery and therapeutic efficacy of nanotherapeutics systems as they need to bypass the airway defense. Targeted autophagy augmentation has emerged, as a promising therapeutic utility for alleviating obstructive lung diseases, and promoting healthy aging. A targeted dendrimer-based approach has been designed to penetrate the airway obstruction and allow the selective correction of proteostasis/autophagy in the diseased cells while circumventing the side effects. This report describes methods for synthesis and therapeutic evaluation of autophagy augmenting dendrimers in the treatment of obstructive lung disease(s). The formulations and methods of autophagy augmentation described here are currently under clinical development in our laboratory for alleviating pathogenesis and progression of chronic obstructive lung diseases, and promoting healthy aging.

Stimuli Responsive Scaffolds Based on Carboxymethyl Starch and Poly(2-Dimethylaminoethyl Methacrylate) for Anti-Inflammatory Drug Delivery.

The purpose of the study is to obtain multicomponent polyelectrolyte hydrogels with optimal synergistic properties by combining a modified starch with a synthetic one. Thus, new low-cost and biocompatible semi-interpenetrating polymer network (semi-IPN) hydrogels of carboxymethyl starch and poly(2-dimethylaminoethyl methacrylate) are prepared and investigated. The synthesized hydrogels are studied with respect to the specific characteristics of the gels: swelling kinetics, thermal analysis, viscoelastic characteristics, and their ability to be used as a matrix in drug delivery systems. Therefore, the semi-IPN gels are loaded with ibuprofen, followed by additional tests to assess the in vitro drug release. The cytocompatibility of the hydrogels with respect to their composition is evaluated in vitro on fibroblast cell culture. The investigations confirm the obtainment of new semi-IPN hydrogels with pH and temperature responsiveness, good mechanical strength, and potential for use as drug delivery systems or transdermal patches.

4-Methyltrityl-Protected Pyrrole and Imidazole Building Blocks for Solid Phase Synthesis of DNA-Binding Polyamides.

DNA-binding polyamides are synthetic oligomers of pyrrole/imidazole units with high specificity and affinity for double-stranded DNA. To increase their synthetic diversity, we report a mild methodology based on 4-methyltrityl (Mtt) solid phase peptide synthesis (SPPS), whose building blocks are more accessible than the standard Fmoc and Boc SPPS ones. We demonstrate the robustness of the approach by preparing and studying a hairpin with all precursors. Importantly, our strategy is orthogonal and compatible with sensitive molecules and could be readily automated.

Biobased chiral semi-crystalline or amorphous high-performance polyamides and their scalable stereoselective synthesis.

The use of renewable feedstock is one of the twelve key principles of sustainable chemistry. Unfortunately, bio-based compounds often suffer from high production cost and low performance. To fully tap the potential of natural compounds it is important to utilize their functionalities that could make them superior compared to fossil-based resources. Here we show the conversion of (+)-3-carene, a by-product of the cellulose industry into ε-lactams from which polyamides. The lactams are selectively prepared in two diastereomeric configurations, leading to semi-crystalline or amorphous, transparent polymers that can compete with the thermal properties of commercial high-performance polyamides. Copolyamides with caprolactam and laurolactam exhibit an increased glass transition and amorphicity compared to the homopolyamides, potentially broadening the scope of standard polyamides. A four-step one-vessel monomer synthesis, applying chemo-enzymatic catalysis for the initial oxidation step, is established. The great potential of the polyamides is outlined.

Facile Synthesis of Poly(DMAEMA-co-MPS)-coated Porous Silica Nanocarriers as Dual-targeting Drug Delivery Platform: Experimental and Biological Investigations.

Inorganic structures with functionalized polymers play essential roles in diverse biological trends. Herein, thermal and CO2 dual-stimuli nanomaterials composed of mesoporous silica nanoparticles (MSN) anchored with two grafted copolymers: poly(3-methacryloxypropyltrimethoxysilane) "PMPS" & poly(N,N-dimethylaminoethyl methacrylate) "PDMAEMA" were synthesized via one-step reaction and characterized by BET as well as BJH methods to estimate pore sizes, pore volumes, and surface areas. The smart PDMAEMA acted as an active gatekeeper to adjust the loading or in vitro release processes of a fungicidal drug-loaded inside the mesopores by altering temperature or CO2 of the tested environment. Furthermore, treating the nanomaterials by CO2 for a few minutes was found to have a bactericidal effect with promising results as indicated by the disk diffusion technique. In general, the positive biological activity against selected strains of bacteria and fungi indicates that these particles may be helpful for engineering more efficient antifungal or antibacterial agents for pharmaceutical applications.

Targeted suppression of metastasis regulatory transcription factor SOX2 in various cancer cell lines using a sequence-specific designer pyrrole-imidazole polyamide.

Metastasis, a deadly feature of cancer, compromises the prognosis and accounts for mortality in the majority of cancer patients. SOX2, a well-known pluripotency transcription factor, plays a central role in cell fate determination and has an overlapping role as a regulatory factor in tumorigenesis and metastasis. The demand is increasing for clinically useful strategies for artificial control of SOX2 expression and its complex transcription machinery in cancer cells. N-Methylpyrrole (Py) and N-methylimidazole (Im) polyamides are small programmable designer ligands that can be pre-programmed to selectively recognize DNA sequence and control endogenous gene expression. Herein, we evaluated the anticancer activity of a designer ligand (SOX2i). SOX2i remarkably altered the expression of SOX2 at the mRNA and protein level in human cancer cell lines such as SW620 (colorectal adenocarcinoma), MKN45 (gastric adenocarcinoma), MCF7 (breast carcinoma), U2OS (osteosarcoma) and other cancer cell lines of different origin and type. Genome-wide transcriptome analysis and cell-based assays showed SOX2 to be a downregulated upstream regulator that alters cell proliferation, cell cycle progression, metabolism and apoptotic pathway. Studies in the mouse model confirmed the anti-metastatic property of SOX2i. SOX2i inhibited the expression of genes associated with EMT and stemness. Moreover, Wnt-canonical signaling was found to be downregulated in the SOX2i-treated group. Our proof-of-concept study supports the potential of DNA-based programmable small molecules for controlling the key regulatory factors associated with tumorigenesis and metastasis.

Modulating DNA by polyamides to regulate transcription factor PU.1-DNA binding interactions.

Hairpin polyamides are synthetic small molecules that bind DNA minor groove sequence-selectively and, in many sequences, induce widening of the minor groove and compression of the major groove. The structural distortion of DNA caused by polyamides has enhanced our understanding of the regulation of DNA-binding proteins via polyamides. Polyamides have DNA binding affinities that are comparable to those proteins, therefore, can potentially be used as therapeutic agents to treat diseases caused by aberrant gene expression. In fact, many diseases are characterized by over- or under-expressed genes. PU.1 is a transcription factor that regulates many immune system genes. Aberrant expression of PU.1 has been associated with the development of acute myeloid leukemia (AML). We have, therefore, designed and synthesized ten hairpin polyamides to investigate their capacity in controlling the PU.1-DNA interaction. Our results showed that nine of the polyamides disrupt PU.1-DNA binding and the inhibition capacity strongly correlates with binding affinity. One molecule, FH1024, was observed forming a FH1024-PU.1-DNA ternary complex instead of inhibiting PU.1-DNA binding. This is the first report of a small molecule that is potentially a weak agonist that recruits PU.1 to DNA. This finding sheds light on the design of polyamides that exhibit novel regulatory mechanisms on protein-DNA binding.