Role of Hydrophobic Modification in Spermine-Based Poly(ß-amino ester)s for siRNA Delivery and Their Spray-Dried Powders for Inhalation and Improved Storage.

After RNAi was first discovered over 20 years ago, siRNA-based therapeutics are finally becoming reality. However, the delivery of siRNA has remained a challenge. In our previous research, we found that spermine-based poly(ß-amino ester)s are very promising for siRNA delivery. However, the role of hydrophobic modification in siRNA delivery of spermine-based poly(ß-amino ester)s is not fully understood yet. In the current work, we synthesized spermine-based poly(ß-amino ester)s with different percentages of oleylamine side chains, named P(SpOABAE). The chemical structures of the polymers were characterized by 1H NMR. The polymers showed efficient siRNA encapsulation determined by SYBR Gold assays. The hydrodynamic diameters of the P(SpOABAE) polyplexes from charge ratio N/P 1 to 20 were 30-100 nm except for aggregation phenomena observed at N/P 3. Morphology of the polyplexes was visualized by atomic force microscopy, and cellular uptake was determined by flow cytometry in H1299 cells, where all the polyplexes showed significantly higher cellular uptake than hyperbranched polyethylenimine (25 kDa). The most hydrophobic P(SpOABAE) polyplexes were able to achieve more than 90% GFP knockdown in H1299/eGFP cells. The fact that gene silencing efficacy increased with hydrophobicity but cellular uptake was affected by both charge and hydrophobic interactions highlights the importance of endosomal escape. For pulmonary administration and improved storage stability, the polyplexes were spray-dried. Results confirmed the maintained siRNA activity after storage for 3 months at room temperature, indicating potential for dry powder inhalation.

Merging σ-Bond Metathesis with Polymerization Catalysis: Insights into Rare-Earth Metal Complexes, End-Group Functionalization, and Application Prospects.

Polymers with well-defined structures, synthesized through metal-catalyzed processes, and having end groups exhibiting different polarity and reactivity than the backbone, are gaining considerable attention in both scientific and industrial communities. These polymers show potential applications as fundamental building blocks and additives in the creation of innovative functional materials. Investigations are directed toward identifying the most optimal and uncomplicated synthetic approach by employing a combination of living coordination polymerization mediated by rare-earth metal complexes and C-H bond activation reaction by σ-bond metathesis. This combination directly yields catalysts with diverse functional groups from a single precursor, enabling the production of terminal-functionalized polymers without the need for sequential reactions, such as termination reactions. The utilization of this innovative methodology allows for precise control over end-group functionalities, providing a versatile approach to tailor the properties and applications of the resulting polymers. This perspective discusses the principles, challenges, and potential advancements associated with this synthetic strategy, highlighting its significance in advancing the interface of metalorganic chemistry, polymer chemistry, and materials science.

Correction to "Cytocompatible Triblock Copolymers with Controlled Microstructure Enabling Orthogonally Functionalized Bio-polymer Conjugates".

[This corrects the article DOI: 10.1021/acs.macromol.3c02238.].

Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis.

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Synthesis and application of spermine-based amphiphilic poly(ß-amino ester)s for siRNA delivery.

Small interfering RNA (siRNA) can trigger RNA interference (RNAi) to therapeutically silence disease-related genes in human cells. The approval of siRNA therapeutics by the FDA in recent years generated a new hope in novel and efficient siRNA therapeutics. However, their therapeutic application is still limited by the lack of safe and efficient transfection vehicles. In this study, we successfully synthesized a novel amphiphilic poly(ß-amino ester) based on the polyamine spermine, hydrophobic decylamine and 1,4-butanediol diacrylate, which was characterized by 1H NMR spectroscopy and size exclusion chromatography (SEC, Mn = 6000 Da). The polymer encapsulated siRNA quantitatively from N/P 5 on as assessed by fluorescence intercalation while maintaining optimal polyplex sizes and zeta potentials. Biocompatibility and cellular delivery efficacy were also higher than those of the commonly used cationic, hyperbranched polymer polyethylenimine (PEI, 25 kDa). Optimized formulations mediated around 90% gene silencing in enhanced green fluorescence protein expressing H1299 cells (H1299-eGFP) as determined by flow cytometry. These results suggest that spermine-based, amphiphilic poly(ß-amino ester)s are very promising candidates for efficient siRNA delivery.

Spermine-Based Poly(ß-amino ester)s for siRNA Delivery against Mutated KRAS in Lung Cancer.

Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(ß-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(ß-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(ß-amino ester)s are promising materials for therapeutic siRNA delivery.

Overcoming the blood-brain barrier? - prediction of blood-brain permeability of hydrophobically modified polyethylenimine polyplexes for siRNA delivery into the brain with in vitro and in vivo models.

The blood-brain barrier (BBB) is a highly selective biological barrier that represents a major bottleneck in the treatment of all types of central nervous system (CNS) disorders. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach, e.g., for brain tumors, by downregulating brain tumor-related genes and inhibiting tumor growth via RNA interference. In an effort to develop efficient siRNA nanocarriers for crossing the BBB, we utilized polyethyleneimine (PEI) polymers hydrophobically modified with either stearic-acid (SA) or dodecylacrylamide (DAA) subunits and evaluated their suitability for delivering siRNA across the BBB in in vitro and in vivo BBB models depending on their structure. Physicochemical characteristics of siRNA-polymer complexes (polyplexes (PXs)), e.g., particle size and surface charge, were measured by dynamic light scattering and laser Doppler anemometry, whereas siRNA condensation ability of polymers and polyplex stability was evaluated by spectrophotometric methods. The composition of the biomolecule corona that absorbs on polyplexes upon encountering physiological fluids was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Cellular internalization abilities of PXs into brain endothelial cells (hCMEC/D3) was confirmed, and a BBB permeation assay using a human induced pluripotent stem cell (hiPSC)-derived BBB model revealed similar abilities to cross the BBB for all formulations under physiological conditions. However, biodistribution studies of radiolabeled PXs in mice were inconsistent with in vitro results as the detected amount of radiolabeled siRNA in the brain delivered with PEI PXs was higher compared to PEI-SA PXs. Taken together, PEI PXs were shown to be a suitable nanocarrier to deliver small amounts of siRNA across the BBB into the brain but more sophisticated human BBB models that better represent physiological conditions and biodistribution are required to provide highly predictive in vitro data for human CNS drug development in the future.

Transferrin-modified chitosan nanoparticles for targeted nose-to-brain delivery of proteins.

Nose-to-brain delivery presents a promising alternative route compared to classical blood-brain barrier passage, especially for the delivery of high molecular weight drugs. In general, macromolecules are rapidly degraded in physiological environment. Therefore, nanoparticulate systems can be used to protect biomolecules from premature degradation. Furthermore, targeting ligands on the surface of nanoparticles are able to improve bioavailability by enhancing cellular uptake due to specific binding and longer residence time. In this work, transferrin-decorated chitosan nanoparticles are used to evaluate the passage of a model protein through the nasal epithelial barrier in vitro. It was demonstrated that strain-promoted azide-alkyne cycloaddition reaction can be utilized to attach a functional group to both transferrin and chitosan enabling a rapid covalent surface-conjugation under mild reaction conditions after chitosan nanoparticle preparation. The intactness of transferrin and its binding efficiency were confirmed via SDS-PAGE and SPR measurements. Resulting transferrin-decorated nanoparticles exhibited a size of about 110-150 nm with a positive surface potential. Nanoparticles with the highest amount of surface bound targeting ligand also displayed the highest cellular uptake into a human nasal epithelial cell line (RPMI 2650). In an air-liquid interface co-culture model with glioblastoma cells (U87), transferrin-decorated nanoparticles showed a faster passage through the epithelial cell layer as well as increased cellular uptake into glioblastoma cells. These findings demonstrate the beneficial characteristics of a specific targeting ligand. With this chemical and technological formulation concept, a variety of targeting ligands can be attached to the surface after nanoparticle formation while maintaining cargo integrity.

Synthesis and Application of Low Molecular Weight PEI-Based Copolymers for siRNA Delivery with Smart Polymer Blends.

Polyethylenimine (PEI) is a commonly used cationic polymer for small-interfering RNA (siRNA) delivery due to its high transfection efficiency at low commercial cost. However, high molecular weight PEI is cytotoxic and thus, its practical application is limited. In this study, different formulations of low molecular weight PEI (LMW-PEI) based copolymers polyethylenimine-g-polycaprolactone (PEI-PCL) (800 Da-40 kDa) and PEI-PCL-PEI (5-5-5 kDa) blended with or without polyethylene glycol-b-polycaprolactone (PEG-PCL) (5 kDa-4 kDa) are investigated to prepare nanoparticles via nanoprecipitation using a solvent displacement method with sizes ≈100 nm. PEG-PCL can stabilize the nanoparticles, improve their biocompatibility, and extend their circulation time in vivo. The nanoparticles composed of PEI-PCL-PEI and PEG-PCL show higher siRNA encapsulation efficiency than PEI-PCL/PEG-PCL based nanoparticles at low N/P ratios, higher cellular uptake, and a gene silencing efficiency of ≈40% as a result of the higher molecular weight PEI blocks. These results suggest that the PEI-PCL-PEI/PEG-PCL nanoparticle system could be a promising vehicle for siRNA delivery at minimal synthetic effort.

Photocatalytic CO2 Conversion Using Metal-Containing Coordination Polymers and Networks: Recent Developments in Material Design and Mechanistic Details.

International guidelines have progressively addressed global warming which is caused by the greenhouse effect. The greenhouse effect originates from the atmosphere's gases which trap sunlight which, as a consequence, causes an increase in global surface temperature. Carbon dioxide is one of these greenhouse gases and is mainly produced by anthropogenic emissions. The urgency of removing atmospheric carbon dioxide from the atmosphere to reduce the greenhouse effect has initiated the development of methods to covert carbon dioxide into valuable products. One approach that was developed is the photocatalytic transformation of CO2. Photocatalysis addresses environmental issues by transferring CO2 into value added chemicals by mimicking the natural photosynthesis process. During this process, the photocatalytic system is excited by light energy. CO2 is adsorbed at the catalytic metal centers where it is subsequently reduced. To overcome several obstacles for achieving an efficient photocatalytic reduction process, the use of metal-containing polymers as photocatalysts for carbon dioxide reduction is highlighted in this review. The attention of this manuscript is directed towards recent advances in material design and mechanistic details of the process using different polymeric materials and photocatalysts.

Engineering Burkholderia sacchari to enhance poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] production from xylose and hexanoate.

Burkholderia sacchari LFM101 LMG19450T is a Brazilian bacterium isolated from sugarcane crops soil and a promising biotechnological platform for bioprocesses. It is an efficient producer of poly(3-hydroxybutyrate) from carbohydrates including xylose. In the present work, the expression of B. sacchari xylose consumption genes (xylA, xylB and tktA) was combined with the expression of Aeromonas sp. phaC (PHA synthase), aiming to increase both the growth rates in xylose and the 3-hydroxyhexanoate (3HHx) molar fractions in the produced PHA. Genes were cloned into pBBR1MCS-2 vectors and then expressed in the B. sacchari PHA- mutant LFM344. Maximum specific growth rates on xylose and PHA accumulation capacity of all recombinants were evaluated. In bioreactor experiments, up to 55.5 % CDW was accumulated as copolymer, hexanoate conversion to 3HHx raised from 2 % to 54 % of the maximum theoretical value, compared to wild type. 3HHx mol% ranged from 8 to 35, and molecular weights were between 111 and 220 kg/mol. Thermal analysis measurement showed a decrease in Tg and Tm values with higher 3HHx fraction, indicating improved thermomechanical characteristics. Recombinants construction and bioreactor strategies allowed the production of P(3HB-co-3HHx) with controlled monomeric composition from xylose and hexanoate, allowing its application in diverse fields, including the medical area.

Impact of crystalline and amorphous matrices on successful spray drying of siRNA polyplexes for inhalation of nano-in-microparticles.

To develop stable and inhalable dry powder formulations with long shelf life, we spray dried polyplexes consisting of siRNA and a polyethylenimine based block copolymer in presence of mannitol or trehalose. We investigated the effect of inlet (T-In) and outlet (T-Out) temperature on the recovery of siRNA as well as adsorption effects within the tubing material. Choosing a low abrasion silicon tubing prevented siRNA loss due to adsorption. Mannitol and trehalose formulations preserved siRNA integrity regardless of excipient concentration and temperature at T-Out below the siRNA melting temperature. Trehalose formulations allowed full siRNA recovery whereas mannitol formulations resulted in spray drying induced losses of ~20 % siRNA and of 50-60 % polymer. Mannitol formulations showed optimal aerodynamic characteristics as confirmed by next generation impaction analysis based upon siRNA content. All spray dried formulations resulted in GFP silencing comparable or better than freshly prepared polyplexes. To test if the observed results could be transferred, formulations of siRNA and transferrin-PEI conjugates were spray dried, characterized and used to transfect primary human T cells ex vivo. Results confirmed successful silencing of the Th2 transcription factor GATA3 in primary CD4+ T cells with spray dried formulations as a potential treatment for severe asthma.

From adsorption to covalent bonding: Apolipoprotein E functionalization of polymeric nanoparticles for drug delivery across the blood-brain barrier.

The blood-brain barrier (BBB) is composed of brain endothelial cells, pericytes, and astrocytes, which build a tight cellular barrier. Therapeutic (macro)molecules are not able to transit through the BBB in their free form. This limitation is bypassed by apolipoprotein E (ApoE)-functionalized polymeric nanoparticles (NPs) that are able to transport drugs (e.g. dalargin, loperamide, doxorubicin, nerve growth factor) across the BBB via low density lipoprotein (LDL) receptor mediated transcytosis. Coating with polysorbate 80 or poloxamer 188 facilitates ApoE adsorption onto polymeric NPs enabling recognition by LDL receptors of brain endothelial cells. This effect is even enhanced when NPs are directly coated with ApoE without surfactant anchor. Similarly, covalent coupling of ApoE to NPs that bear reactive groups on their surface leads to significantly improved brain uptake while avoiding the use of surfactants. Several in vitro BBB models using brain endothelial cells or co-cultures with astrocytes/pericytes/glioma cells are described which provide first insights regarding the ability of a drug delivery system to cross this barrier. In vivo models are employed to simulate central nervous system-relevant diseases such as Alzheimer's or Parkinson's disease and cerebral cancer.

The Impact of Nylon-3 Copolymer Composition on the Efficiency of siRNA Delivery to Glioblastoma Cells.

Glioblastoma multiforme is a devastating disease that has attracted enormous attention due to poor prognosis and high recurrence. Small interfering RNA (siRNA) in principle offers a promising therapeutic approach by the downregulation of disease-related genes via RNA interference. For efficient siRNA delivery to target sites, cationic polymers are often used in preclinical studies for the protection of siRNA and complex formation based on electrostatic interactions. In an effort to develop biocompatible and efficient nanocarriers with a translational outlook for optimal gene silencing at reduced toxicity, we synthesized two sets of nylon-3 copolymers with variable cationic content (DM or NM monomer) and hydrophobic subunits (CP monomer) and evaluated their suitability for in vitro siRNA delivery into glioblastoma cells. DM0.4/CP0.6 and NM0.4/CP0.6 polymers with similar subunit ratios were synthesized to compare the effect of different cationic subunits. Additionally, we utilized NM0.2/CP0.8 polymers to evaluate the impact of the different hydrophobic content in the polymer chain. The siRNA condensation ability and polymer-siRNA complex stability was evaluated by unmodified and modified SYBR gold assays, respectively. Further physicochemical characteristics, e.g., particle size and surface charge, were evaluated by dynamic light scattering and laser Doppler anemometry, whereas a relatively new method for polyplex size distribution analysis-tunable resistive pulse sensing-was additionally developed and compared to DLS measurements. Transfection efficiencies, the route of cell internalization, and protein knockdown abilities in glioblastoma cells were investigated by flow cytometry. Furthermore, cellular tolerability was evaluated by MTT and LDH assays. All the polymers efficiently condensed siRNA at N/P ratios of three, whereas polymers with NM cationic subunits demonstrated smaller particle size and lower polyplex stability. Furthermore, NM0.2/CP0.8 polyplexes with the highest hydrophobic content displayed significantly higher cellular internalization in comparison to more cationic formulations and successful knockdown capabilities. Detailed investigations of the cellular uptake route demonstrated that these polyplexes mainly follow clathrin-mediated endocytotic uptake mechanisms, implying high interaction capacity with cellular membranes. Taken together with conducive toxicity profiles, highly hydrophobic nylon-3 polymers provide an appropriate siRNA delivery agent for the potential treatment of glioblastoma.

Metal-Catalyzed Group-Transfer Polymerization: A Versatile Tool for Tailor-Made Functional (Co)Polymers.

Accommodating the increasing demand for tailor-made polymers is a major goal in polymer chemistry. Therefore, the investigation of polymerization techniques, which allow the precise synthesis of macromolecules is of exceptional interest. Ionic or controlled radical polymerization are capable living-type methods for the generation of uniform polymers. However, even these approaches reach their limits in certain issues. In the last decades, group-transfer polymerization (GTP) and especially metal-catalyzed GTP have proven to give access to a plethora of tailor-made homo- and copolymers based on α,ß-unsaturated monomers. Thereby, GTP has established its potential in the development of functional and smart polymers. This concept article highlights the most significant progress in metal-catalyzed GTP with a focus on functional (co)polymers including different polymeric architectures and microstructures.

Toolbox of Nonmetallocene Lanthanides: Multifunctional Catalysts in Group-Transfer Polymerization.

Herein, we present a fundamental study of isostructural 2-methoxyethylamino-bis(phenolate)-lanthanide complexes [(ONOO)RM(X)(THF)] (M = Lu, Y; R = tBu, CMe2Ph, X = CH2TMS, collidine; THF = tetrahydrofuran; TMS = trimethylsilyl) for rare-earth metal-mediated group-transfer polymerization (GTP). This analysis includes the differentiation of electron-donating and nondonating vinyl monomers and two metal centers with regard to the ionic radius (yttrium and lutetium). In addition, highly nucleophilic alkyl initiators are compared with electron-donating heteroaromatic initiators. Our examinations include the impact of these parameters on the activity, initiator efficiency, and tacticity of the obtained polymers. Density functional theory calculations and proposed catalyst structure determinations via X-ray analysis support these investigations. This facilitates the selection of the best metal and initiator combination to address efficient and stereospecific polymerization of a broad range of Michael monomers. [(ONOO)tBuLu(X)(THF)] shows the highest activity of 2220 h-1 (normalized turnover frequency) for the polymerization of 2-vinylpyridine due to the higher Lewis-acidity of lutetium. Through C(sp3)-H bond activation, catalysts with higher initiator efficiency in N,N'-dimethylacrylamide (DMAA) and diethylvinylphosphonate polymerization were synthesized. Remarkably, [(ONOO)tBuY(collidine)(THF)] was capable of stereospecifically polymerizing DMAA to highly isotactic poly(DMAA) (Pm = 0.94). Overall, the kinetics studies reveal a living-type GTP mechanism for all of the tested catalysts, enabling precise molecular-weight predeterminations with narrow molecular weight distributions (D ≤ 1.06).

CO2-Controlled One-Pot Synthesis of AB, ABA Block, and Statistical Terpolymers from ß-Butyrolactone, Epoxides, and CO2.

Terpolymerizations of (rac)-ß-butyrolactone (BBL), cyclohexene oxide (CHO), and carbon dioxide were realized in one-pot reactions utilizing a Lewis acid BDICF3-Zn-N(SiMe3)2 (1) catalyst. The type of polymerization can be regulated and switched between ring-opening polymerization (ROP) of BBL and CHO/CO2 copolymerization by the presence of CO2 in the reaction mixture. Applying 3 bar CO2 to the three-component system leads to similar reaction rates for copolymerization and ROP and therefore to a terpolymer with a statistical composition, whereas 40 bar CO2 affords exclusive copolymerization of CHO/CO2. Two-dimensional NMR spectroscopy and polarimetry provided a deeper understanding of the underlying mechanism. Furthermore, copolymerization of cyclopentene oxide (CPO) and CO2 was performed, resulting in the highest reported TOF of 3200 h-1 together with 99% polycarbonate selectivity. Terpolymerizations of CPO/CO2 and BBL were successfully conducted using the established reaction pathways.

Next Generation Multiresponsive Nanocarriers for Targeted Drug Delivery to Cancer Cells.

C-H bond activation of 2-methoxyethylamino-bis(phenolate)-yttrium catalysts allowed the synthesis of BAB block copolymers comprised of 2-vinylpyridine (2VP; monomer A) and diethylvinylphosphonate (DEVP; monomer B) as the A and B blocks, respectively, by rare-earth-metal-mediated group-transfer polymerization (REM-GTP). The inherent multi-stimuli-responsive character and drug-loading and -release capabilities were observed to be dependent on the chain length and monomer ratios. Cytotoxicity assays revealed the biocompatibility and nontoxic nature of the obtained micelles toward ovarian cancer (HeLa) cells. The BAB block copolymers effectively encapsulated, transported, and released doxorubicin (DOX) within HeLa cells. REM-GTP enables access to previously unattainable vinylphosphonate copolymer structures, and thereby unlocks their full potential as nanocarriers for stimuli-responsive drug delivery in HeLa cells. The self-evident consequence is the application of these new micelles as potent drug-delivery vehicles with reduced side effects in future cancer therapies.