Optimization of Mixed Micelles Based on Oppositely Charged Block Copolymers by Machine Learning for Application in Gene Delivery.

The COVID-19 mRNA vaccines represent a milestone in developing non-viral gene carriers, and their success highlights the crucial need for continued research in this field to address further challenges. Polymer-based delivery systems are particularly promising due to their versatile chemical structure and convenient adaptability, but struggle with the toxicity-efficiency dilemma. Introducing anionic, hydrophilic, or "stealth" functionalities represents a promising approach to overcome this dilemma in gene delivery. Here, two sets of diblock terpolymers are created comprising hydrophobic poly(n-butyl acrylate) (PnBA), a copolymer segment made of hydrophilic 4-acryloylmorpholine (NAM), and either the cationic 3-guanidinopropyl acrylamide (GPAm) or the 2-carboxyethyl acrylamide (CEAm), which is negatively charged at neutral conditions. These oppositely charged sets of diblocks are co-assembled in different ratios to form mixed micelles. Since this experimental design enables countless mixing possibilities, a machine learning approach is applied to identify an optimal GPAm/CEAm ratio for achieving high transfection efficiency and cell viability with little resource expenses. After two runs, an optimal ratio to overcome the toxicity-efficiency dilemma is identified. The results highlight the remarkable potential of integrating machine learning into polymer chemistry to effectively tackle the enormous number of conceivable combinations for identifying novel and powerful gene transporters.

Optimizing Biocompatibility and Gene Delivery with DMAEA and DMAEAm: A Niacin-Derived Copolymer Approach.

Gene therapy is pivotal in nanomedicine, offering a versatile approach to disease treatment. This study aims to achieve an optimal balance between biocompatibility and efficacy, which is a common challenge in the field. A copolymer library is synthesized, incorporating niacin-derived monomers 2-acrylamidoethyl nicotinate (AAEN) or 2-(acryloyloxy)ethyl nicotinate (AEN) with N,N-(dimethylamino)ethyl acrylamide (DMAEAm) or hydrolysis-labile N,N-(dimethylamino)ethyl acrylate (DMAEA). Evaluation of the polymers' cytotoxicity profiles reveals that an increase in AAEN or DMAEA molar ratios correlates with improved biocompatibility. Remarkably, an increase in AAEN in both DMAEA and DMAEAm copolymers demonstrated enhanced transfection efficiencies of plasmid DNA in HEK293T cells. Additionally, the top-performing polymers demonstrate promising gene expression in challenging-to-transfect cells (THP-1 and Jurkat cells) and show no significant effect on modulating immune response induction in ex vivo treated murine monocytes. Overall, the best performing candidates exhibit an optimal balance between biocompatibility and efficacy, showcasing potential advancements in gene therapy.

Improving Gene Delivery: Synergy between Alkyl Chain Length and Lipoic Acid for PDMAEMA Hydrophobic Copolymers.

In the field of gene delivery, hydrophobic cationic copolymers hold great promise. They exhibit improved performance by effectively protecting genetic material from serum interactions while facilitating interactions with cellular membranes. However, managing cytotoxicity remains a significant challenge, prompting an investigation into suitable hydrophobic components. A particularly encouraging approach involves integrating nutrient components, like lipoic acid, which is known for its antioxidant properties and diverse cellular benefits such as cellular metabolism and growth. In this study, a copolymer library comprising 2-(dimethylamino)ethyl methacrylate (DMAEMA) and lipoic acid methacrylate (LAMA), combined with either n-butyl methacrylate (nBMA), ethyl methacrylate (EMA), or methyl methacrylate (MMA), is synthesized. This enables to probe the impact of lipoic acid incorporation while simultaneously exploring the influence of pendant acyclic alkyl chain length. The inclusion of lipoic acid results in a notable boost in transfection efficiency  while maintaining low cytotoxicity. Interestingly, higher levels of transfection efficiency are achieved in the presence of nBMA, EMA, or MMA. However, a positive correlation between pendant acyclic alkyl chain length and cytotoxicity is observed. Consequently, P(DMAEMA-co-LAMA-co-MMA), emerges as a promising candidate. This is attributed to the optimal combination of low cytotoxic MMA and transfection-boosting LAMA, highlighting the crucial synergy between LAMA and MMA.

Stimuli-Responsive Non-viral Nanoparticles for Gene Delivery.

Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.

Efficient Gene Delivery of Tailored Amphiphilic Polypeptides by Polyplex Surfing.

Within this study, an amphiphilic and potentially biodegradable polypeptide library based on poly[(4-aminobutyl)-l-glutamine-stat-hexyl-l-glutamine] [P(AB-l-Gln-stat-Hex-l-Gln)] was investigated for gene delivery. The influence of varying proportions of aliphatic and cationic side chains affecting the physicochemical properties of the polypeptides on transfection efficiency was investigated. A composition of 40 mol% Hex-l-Gln and 60 mol % AB-l-Gln (P3) was identified as best performer over polypeptides with higher proportions of protonatable monomers. Detailed studies of the transfection mechanism revealed the strongest interaction of P3 with cell membranes, promoting efficient endocytic cell uptake and high endosomal release. Spectrally, time-, and z-resolved fluorescence microscopy further revealed the crucial role of filopodia surfing in polyplex-cell interaction and particle internalization in lamellipodia regions, followed by rapid particle transport into cells. This study demonstrates the great potential of polypeptides for gene delivery. The amphiphilic character improves performance over cationic homopolypeptides, and the potential biodegradability is advantageous toward other synthetic polymeric delivery systems.

pH-sensitive packaging of cationic particles by an anionic block copolymer shell.

Cationic non-viral vectors show great potential to introduce genetic material into cells, due to their ability to transport large amounts of genetic material and their high synthetic versatility. However, designing materials that are effective without showing toxic effects or undergoing non-specific interactions when applied systemically remains a challenge. The introduction of shielding polymers such as polyethylene glycol (PEG) can enhance biocompatibility and circulation time, however, often impairs transfection efficiency. Herein, a multicomponent polymer system is introduced, based on cationic and hydrophobic particles (P(nBMA46-co-MMA47-co-DMAEMA90), (PBMD)) with high delivery performance and a pH-responsive block copolymer (poly((N-acryloylmorpholine)-b-(2-(carboxy)ethyl acrylamide)) (P(NAM72-b-CEAm74), PNC)) as shielding system, with PNAM as alternative to PEG. The pH-sensitive polymer design promotes biocompatibility and excellent stability at extracellular conditions (pH 7.4) and also allows endosomal escape and thus high transfection efficiency under acidic conditions. PNC shielded particles are below 200 nm in diameter and showed stable pDNA complexation. Further, interaction with human erythrocytes at extracellular conditions (pH 7.4) was prevented, while acidic conditions (pH 6) enabled membrane leakage. The particles demonstrate transfection in adherent (HEK293T) as well as difficult-to-transfect suspension cells (K-562), with comparable or superior efficiency compared to commercial linear poly(ethylenimine) (LPEI). Besides, the toxicity of PNC-shielded particles was significantly minimized, in particular in K-562 cells and erythrocytes. In addition, a pilot in vivo experiment on bone marrow blood cells of mice that were injected with PNC-shielded particles, revealed slightly enhanced cell transfection in comparison to naked pDNA. This study demonstrates the applicability of cationic hydrophobic polymers for transfection of adherent and suspension cells in culture as well as in vivo by co-formulation with pH-responsive shielding polymers, without substantially compromising transfection performance.

The impact of anionic polymers on gene delivery: how composition and assembly help evading the toxicity-efficiency dilemma.

Cationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymerization. The copolymer poly[(n-butyl acrylate)-b-(2-(dimethyl amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphology, interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addition of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.

Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles.

Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

Improved Bioactivity of the Natural Product 5-Lipoxygenase Inhibitor Hyperforin by Encapsulation into Polymeric Nanoparticles.

Hyperforin, a highly hydrophobic prenylated acylphloroglucinol from the medical plant St. John's Wort, possesses anti-inflammatory properties and suppresses the formation of proinflammatory leukotrienes by inhibiting the key enzyme 5-lipoxygenase (5-LO). Despite its strong effectiveness and the unique molecular mode of interference with 5-LO, the high lipophilicity of hyperforin hampers its efficacy in vivo and, thus, impairs its therapeutic value, especially because of poor water solubility and strong plasma (albumin) protein binding. To overcome these hurdles that actually apply to many other hydrophobic 5-LO inhibitors, we have encapsulated hyperforin into nanoparticles (NPs) consisting of acetalated dextran (AcDex) to avoid plasma protein binding and thus improve its cellular supply under physiologically relevant conditions. Encapsulated hyperforin potently suppressed 5-LO activity in human neutrophils, but it failed to interfere with 5-LO activity in a cell-free assay, as expected. In the presence of human serum albumin (HSA), hyperforin was unable to inhibit cellular 5-LO activity, seemingly because of strong albumin binding. However, when encapsulated into NPs, hyperforin caused strong inhibition of 5-LO activity in the presence of HSA. Together, encapsulation of the highly hydrophobic hyperforin as a representative of lipophilic 5-LO inhibitors into AcDex-based NPs allows for efficient inhibition of 5-LO activity in neutrophils in the presence of albumin because of effective uptake and circumvention of plasma protein binding.

Beyond Gene Transfection with Methacrylate-Based Polyplexes-The Influence of the Amino Substitution Pattern.

Methacrylate-based polymers represent promising nonviral gene delivery vectors, since they offer a large variety of polymer architectures and functionalities, which are beneficial for specific demands in gene delivery. In combination with controlled radical polymerization techniques, such as the reversible addition-fragmentation chain transfer polymerization, the synthesis of well-defined polymers is possible. In this study we prepared a library of defined linear polymers based on (2-aminoethyl)-methacrylate (AEMA), N-methyl-(2-aminoethyl)-methacrylate (MAEMA), and N,N-dimethyl-(2-aminoethyl)-methacrylate (DMAEMA) monomers, bearing pendant primary, secondary, and tertiary amino groups, and investigated the influence of the substitution pattern on their gene delivery capability. The polymers and the corresponding plasmid DNA complexes were investigated regarding their physicochemical characteristics, cytocompatibility, and transfection performance. The nonviral transfection by methacrylate-based polyplexes differs significantly from poly(ethylene imine)-based polyplexes, as a successful transfection is not affected by the buffer capacity. We observed that polyplexes containing a high content of primary amino groups (AEMA) offered the highest transfection efficiency, whereas polyplexes bearing tertiary amino groups (DMAEMA) exhibited the lowest transfection efficiency. Further insights into the uptake and release mechanisms could be identified by fluorescence and transmission electron microscopy, emphasizing the theory of membrane-pore formation for the time-efficient endosomal release of methacrylate-based vectors.

The Power of Shielding: Low Toxicity and High Transfection Performance of Cationic Graft Copolymers Containing Poly(2-oxazoline) Side Chains.

We show the potential of oligo(2-ethyl-2-oxazoline) (Oxn)-shielded graft copolymers of (2-aminoethyl)-methacrylate and N-methyl-(2-aminoethyl)-methacrylate for pDNA delivery in HEK cells. For the effect of grafting density and side chain length concerning improved transfection properties through the concept of shielding to be investigated, copolymers were synthesized via the macromonomer method using a combination of cationic ring opening polymerization and reversible addition-fragmentation chain transfer polymerization to vary the degree of grafting (DG = 10 and 30%) as well as the side chain degree of polymerization (DP = 5 and 20). Investigations of the polyplex formation, in vitro flow cytometry, and confocal laser scanning microscopy measurements on the copolymer library revealed classical shielding properties of the Ox side chains, including highly reduced cytotoxicity and a partial decrease in transfection efficiency, as also reported for polyethylene glycol shielding. In terms of the transfection efficiency, the best performing copolymers (A- g-Ox5(10) and M- g-Ox5(10)) revealed equal or better performances compared to those of the corresponding homopolymers. In particular, the graft copolymers with low DG and side chain DP transfected well with over 10-fold higher IC50 values. In contrast, a DG of 30% resulted in a loss of transfection efficiency due to missing ability for endosomal release, and a side chain DP of 20 hampered the cellular uptake.

How To Tune the Gene Delivery and Biocompatibility of Poly(2-(4-aminobutyl)-2-oxazoline) by Self- and Coassembly.

Despite their promising potential in gene transfection, the toxicity and limited efficiency of cationic polymers as nonviral vectors are major obstacles for their broader application. The large amount of cationic charges, for example, in poly(ethylene imine) (PEI) is known to be advantageous in terms of their transfection efficiency but goes hand-in-hand with a high toxicity. Consequently, an efficient shielding of the charges is required to minimize toxic effects. In this study, we use a simple mixed-micelle approach to optimize the required charge density for efficient DNA complex formation and to minimize toxicity by using a biocompatible polymer. In detail, we coassembled mixed poly(2-oxazoline) nanostructures ( d ≈ 100 nm) consisting of a hydrophobic-cationic block copolymer (P(NonOx52- b-AmOx184)) and a hydrophobic-hydrophilic stealth block copolymer (P(EtOx155- b-NonOx76) in ratios of 0, 20, 40, 60, 80, and 100 wt % P(NonOx52- b-AmOx184). All micelles with cationic polymers exhibited a very good DNA binding efficiency and dissociation ability, while the bio- and hemocompatibility improved with increasing EtOx content. Analytics via confocal laser scanning microscopy and flow cytometry showed an enhanced cellular uptake, transfection ability, and biocompatibility of all prepared micelleplexes compared to AmOx homopolymers. Micelleplexes with 80 or 100 wt % revealed a similar transfection efficiency as PEI, while the cell viability was significantly higher (80 to 90% compared to 60% for PEI).

Photocontrolled Release of Chemicals from Nano- and Microparticle Containers.

A benzoin-derived diol linker was synthesized and used to generate biocompatible polyesters that can be fully decomposed on demand upon UV irradiation. Extensive structural optimization of the linker unit was performed to enable the defined encapsulation of diverse organic compounds in the polymeric structures and allow for a well-controllable polymer cleavage process. Selective tracking of the release kinetics of encapsulated model compounds from the polymeric nano- and microparticle containers was performed by confocal laser scanning microscopy in a proof-of-principle study. The physicochemical properties of the incorporated and released model compounds ranged from fully hydrophilic to fully hydrophobic. The demonstrated biocompatibility of the utilized polyesters and degradation products enables their use in advanced applications, for example, for the smart packaging of UV-sensitive pharmaceuticals, nutritional components, or even in the area of spatially selective self-healing processes.

Polymersomes with Endosomal pH-Induced Vesicle-to-Micelle Morphology Transition and a Potential Application for Controlled Doxorubicin Delivery.

In order to obtain a novel, pH responsive polymersome system, a series of pH responsive block copolymers were synthesized via the reversible addition-fragmentation chain transfer (RAFT) polymerization of 3,4-dihydro-2H-pyran (DHP) protected 2-hydroxyethyl methacrylate (HEMA) (2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl methacrylate (THP-HEMA)) and 2-(dimethylamino) ethyl methacrylate (DMAEMA) using p(THP-HEMA) as a macro chain transfer agent (mCTA). The degree of polymerization (DP) of the p(THP-HEMA) block was fixed to 35, whereas the DP of the p(DMAEMA) block was systematically varied from 21 to 50. In aqueous solution, the block copolymer with the shortest p(DMAEMA) block (DP = 21) self-assembled into vesicles, while the polymer with 30 units of p(DMAEMA) formed a mixture of micelles and vesicles. The polymer with the longest p(DMAEMA) block (DP = 50) formed exclusively micelles. The corresponding polymersomes exhibited a morphology transition from vesicles at neutral pH values to micelles upon lowering the pH value down to endosomal pH value as investigated by DLS and cryo-TEM. The capability of polymersomes to encapsulate both hydrophobic (e.g., Nile Red) and hydrophilic (e.g., doxorubicin hydrochloride (DOX·HCl)) cargos was verified by in vitro studies. Drug release studies demonstrated that the DOX·HCl release is significantly accelerated under acidic pH values compared to physiological conditions. Cytotoxicity studies revealed that DOX·HCl loaded polymersomes exhibited an efficient cell death comparable to free DOX·HCl. CLSM and flow cytometry studies showed that DOX·HCl loaded vesicles were easily taken up by L929 cells and were mainly located in the cytoplasm and cell nuclei.

Asymmetric flow field-flow fractionation in the field of nanomedicine.

Asymmetric flow field-flow fractionation (AF4) is a widely used and versatile technique in the family of field-flow fractionations, indicated by a rapidly increasing number of publications. It represents a gentle separation and characterization method, where nonspecific interactions are reduced to a minimum, allows a broad separation range from several nano- up to micrometers and enables a superior characterization of homo- and heterogenic systems. In particular, coupling to multiangle light scattering provides detailed access to sample properties. Information about molar mass, polydispersity, size, shape/conformation, or density can be obtained nearly independent of the used material. In this Perspective, the application and progress of AF4 for (bio)macromolecules and colloids, relevant for "nano" medical and pharmaceutical issues, will be presented. The characterization of different nanosized drug or gene delivery systems, e.g., polymers, nanoparticles, micelles, dendrimers, liposomes, polyplexes, and virus-like-particles (VLP), as well as therapeutic relevant proteins, antibodies, and nanoparticles for diagnostic usage will be discussed. Thereby, the variety of obtained information, the advantages and pitfalls of this emerging technique will be highlighted. Additionally, the influence of different fractionation parameters in the separation process is discussed in detail. Moreover, a comprehensive overview is given, concerning the investigated samples, fractionation parameters as membrane types and buffers used as well as the chosen detectors and the corresponding references. The perspective ends up with an outlook to the future.

Tailoring Gene Transfer Efficacy through the Arrangement of Cationic and Anionic Blocks in Triblock Copolymer Micelles.

The arrangement of charged segments in triblock copolymer micelles affects the gene delivery potential of polymeric micelles and can increase the level of gene expression when an anionic segment is incorporated in the outer shell. Triblock copolymers were synthesized by RAFT polymerzation with narrow molar mass distributions and assembled into micelles with a hydrophobic core from poly(n-butyl acrylate). The ionic shell contained either (i) an anionic segment followed by a cationic segment (HAC micelles) or (ii) a cationic block followed by an anionic block (HCA micelles). The pH-responsive anionic block contained 2-carboxyethyl acrylamide (CEAm), while the cationic block comprised 3-guanidinopropyl acrylamide (GPAm). Increasing the molar content of CEAm in HAC and HCA micelles from 6 to 13 mol % improved cytocompatibility and the endosomal escape property, while the HCA micelle with the highest mol % of anionic charges in the outer shell exhibited the highest gene expression. It became evident that improved membrane interaction of the best performing HCA micelle contributed to achieving high gene expression.

Amino-Acid-Derived Anionic Polyacrylamides with Tailored Hydrophobicity-Physicochemical Properties and Cellular Interactions.

Polyanions can internalize into cells via endocytosis without any cell disruption and are therefore interesting materials for biomedical applications. In this study, amino-acid-derived polyanions with different alkyl side-chains are synthesized via postpolymerization modification of poly(pentafluorophenyl acrylate), which is synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization, to obtain polyanions with tailored hydrophobicity and alkyl branching. The success of the reaction is verified by size-exclusion chromatography, NMR spectroscopy, and infrared spectroscopy. The hydrophobicity, surface charge, and pH dependence are investigated in detail by titrations, high-performance liquid chromatography, and partition coefficient measurements. Remarkably, the determined pK a-values for all synthesized polyanions are very similar to those of poly(acrylic acid) (pK a = 4.5), despite detectable differences in hydrophobicity. Interactions between amino-acid-derived polyanions with L929 fibroblasts reveal very slow cell association as well as accumulation of polymers in the cell membrane. Notably, the more hydrophobic amino-acid-derived polyanions show higher cell association. Our results emphasize the importance of macromolecular engineering toward ideal charge and hydrophobicity for polymer association with cell membranes and internalization. This study further highlights the potential of amino-acid-derived polymers and the diversity they provide for tailoring properties toward drug delivery applications.

Vitamin B3 Containing Polymers for Nanodelivery.

Polymeric nanoparticles (NPs) with an integrated dual delivery system enable the controlled release of bioactive molecules and drugs, providing therapeutic advantages. Key design targets include high biocompatibility, cellular uptake, and encapsulating efficiency. In this study, a polymer library derived from niacin, also known as vitamin B3 is synthesized. The library comprises poly(2-(acryloyloxy)ethyl nicotinate) (PAEN), poly(2-acrylamidoethyl nicotinate) (PAAEN), and poly(N-(2-acrylamidoethyl)nicotinamide) (PAAENA), with varying hydrophilicity in the backbone and pendant group linker. All polymers are formulated, and those with increased hydrophobicity yield NPs with homogeneous spherical distribution and diameters below 150 nm, as confirmed by scanning electron microscopy and dynamic light scattering. Encapsulation studies utilizing a model drug, neutral lipid orange (NLO), reveal the influence of polymer backbone on encapsulation efficiency. Specifically, efficiencies of 46% and 96% are observed with acrylate and acrylamide backbones, respectively. Biological investigations showed that P(AEN) and P(AAEN) NPs are non-toxic up to 300 µg mL-1, exhibit superior cellular uptake, and boost cell metabolic activity. The latter is attributed to the cellular release of niacin, a precursor to nicotinamide adenine dinucleotide (NAD), a central coenzyme in metabolism. The results underline the potential of nutrient-derived polymers as pro-nutrient and drug-delivery materials.

Modification of Branched Poly(ethylene imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells.

Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d-fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru-bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weights, degrees of substitution, and linker positions on d-fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10 kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted knockdown of green fluorescent protein (GFP) in triple-negative breast cancer cells (MDA-MB-231) compared to noncancer cells (HEK293T).

Efficient Transfection via an Unexpected Mechanism by Near Neutral Polypiperazines with Tailored Response to Endosomal pH.

Cationic pH-responsive polymers promise to overcome critical challenges in cellular delivery. Ideally, the polymers become selectively charged along the endosomal pathway disturbing only the local membrane and avoiding unintended interactions or cytotoxic side effects at physiological conditions. Polypiperazines represent a novel, hydrophilic class of pH-responsive polymers whose response can be tuned within the relevant pH range (5-7.4). The authors discovered that the polypiperazines are effectively binding plasmid DNA (pDNA) and demonstrate high efficiency in transfection. By design of experiments (DoE), a wide parameter space (pDNA and polymer concentration) is screened to identify the range of effective concentrations for transfection. An isopropyl modified polypiperazine is highly efficient over a wide range of concentrations outperforming linear polyethylenimine (l-PEI, 25 kDa) in regions of low N*/P ratios. A quantitative polymerase chain reaction (qPCR) surprisingly revealed that the pDNA within the piperazine-based polyplexes can be amplified in contrast to polyplexes based on l-PEI. The pDNA must therefore be more accessible and bound differently than for other known transfection polymers. Considering the various opportunities to further optimize their structure, polypiperazines represent a promising platform for designing effective soluble polymeric vectors, which are charge-neutral at physiological conditions.