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.

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.

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.

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.

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.

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.

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.

Tracking the Endosomal Escape: A Closer Look at Calcein and Related Reporters.

Crossing the cellular membrane and delivering active pharmaceuticals or biologicals into the cytosol of cells is an essential step in the development of nanomedicines. One of the most important intracellular processes regarding the cellular uptake of biologicals is the endolysosomal pathway. Sophisticated nanocarriers are developed to overcome a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays are established analyzing the performance of these nanocarriers. Among them, the release of the membrane-impermeable dye calcein has become a popular and straightforward method. It is accessible for most researchers worldwide, allows for rapid conclusions about the release potential, and enables the study of release mechanisms. This review is intended to provide an overview and guidance for scientists applying the calcein release assay. It comprises a survey of several applications in the study of endosomal escape, considerations of potential pitfalls, challenges, and limitations of the assay, and a brief summary of complementary methods. Based on this review, it is hoped to encourage further research groups to take advantage of the calcein release assay for their own purposes and help to create a database for more efficient cross-correlations between nanocarriers.

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.

Nanoparticle sizing in the field of nanomedicine: Power of an analytical ultracentrifuge.

Hydrodynamic and light scattering methods are urgently required for accurate characterization of nanoparticles (NPs) in the field of nanomedicine to unveil their sizes and distributions. A fundamental characterization approach in the field of nanomedicines is, next to standard batch dynamic light scattering (DLS) and increasingly more applied (asymmetrical flow) field-flow fractionation (FFF) coupled to multi-angle laser light scattering (MALLS), the utilization of an analytical ultracentrifuge (AUC). Here, we demonstrate the power of an AUC in comparison to batch DLS and FFF-MALLS to decipher, in detail, the size and dispersity of pharma-relevant, commercial and in-house prepared soft matter NPs, suitable for life science applications. In this study, size and dispersity of poly(lactic-co-glycolic acid) (PLGA) NPs and in-house prepared NPs, consisting of the commercially available pharmapolymer Eudragit® E or of a polymer of similar composition synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization, were investigated. Simultaneously, an insight on the presence of the utilized surfactant on the NP formulations, which is usually limited with other techniques, could be achieved by multi-speed experiments with the AUC in one experimental setting. While the repeatability and ruggedness of observations with modern AUC instruments of the newest generation is demonstrated, the results are further underpinned by the classical relations of hydrodynamics. Investigations aiming at hydrodynamic diameters (from DLS) and radii of gyration (from FFF-MALLS) are critically discussed and compared to the repeatable and rugged investigations by an AUC. The latter is proven to provide a self-sufficient experimental approach for NP characterization in the field of nanomedicine based on absolute principles, compares well to FFF-MALLS, and can unravel issues in NP sizing that arise when more common techniques, such as DLS, are used.

Intracellularly Released Cholesterol from Polymer-Based Delivery Systems Alters Cellular Responses to Pneumolysin and Promotes Cell Survival.

Cholesterol is highly abundant within all human body cells and modulates critical cellular functions related to cellular plasticity, metabolism, and survival. The cholesterol-binding toxin pneumolysin represents an essential virulence factor of Streptococcus pneumoniae in establishing pneumonia and other pneumococcal infections. Thus, cholesterol scavenging of pneumolysin is a promising strategy to reduce S. pneumoniae induced lung damage. There may also be a second cholesterol-dependent mechanism whereby pneumococcal infection and the presence of pneumolysin increase hepatic sterol biosynthesis. Here we investigated a library of polymer particles varying in size and composition that allow for the cellular delivery of cholesterol and their effects on cell survival mechanisms following pneumolysin exposure. Intracellular delivery of cholesterol by nanocarriers composed of Eudragit E100-PLGA rescued pneumolysin-induced alterations of lipid homeostasis and enhanced cell survival irrespective of neutralization of pneumolysin.

Self-assembled PEGylated amphiphilic polypeptides for gene transfection.

In the present study, three biodegradable block copolymers composed of a poly(ethylene glycol) block and a copolypeptide block with varying compositions of cationic L-lysine (L-Lys) and hydrophobic benzyl-L-glutamate (Bzl-L-Glu) were designed for gene delivery applications. The polypeptides were synthesized by ring opening polymerization (ROP) and after orthogonal deprotection of Boc-L-Lys side chains, the polymer exhibited an amphiphilic character. To bind or encapsulate plasmid DNA (pDNA), different formulations were investigated: a nanoprecipitation and an emulsion technique using various organic solvents as well as an aqueous pH-controlled formulation method. The complex and nanoparticle (NP) formations were monitored by dynamic light scattering (DLS), and pDNA interaction was shown by gel electrophoresis and subsequent controlled release with heparin. The polypeptides were further tested for their cytotoxicity as well as biodegradability. The complexes and NPs presenting the most promising size distributions and pDNA binding ability were subsequently evaluated for their transfection efficiency in HEK293T cells. The highest transfection efficiencies were obtained with an aqueous formulation of the polypeptide containing the highest L-Lys content and lowest proportion of hydrophobic, helical structures (P1*), which is therefore a promising candidate for efficient gene delivery by biodegradable gene delivery vectors.

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.

Core-crosslinked, temperature- and pH-responsive micelles: design, physicochemical characterization, and gene delivery application.

Stimuli-responsive block copolymer micelles can provide tailored properties for the efficient delivery of genetic material. In particular, temperature- and pH-responsive materials are of interest, since their physicochemical properties can be easily tailored to meet the requirements for successful gene delivery. Within this study, a stimuli-responsive micelle system for gene delivery was designed based on a diblock copolymer consisting of poly(N,N-diethylacrylamide) (PDEAm) as a temperature-responsive segment combined with poly(aminoethyl acrylamide) (PAEAm) as a pH-responsive, cationic segment. Upon temperature increase, the PDEAm block becomes hydrophobic due to its lower critical solution temperature (LCST), leading to micelle formation. Furthermore, the monomer 2-(pyridin-2-yldisulfanyl)ethyl acrylate (PDSAc) was incorporated into the temperature-responsive PDEAm building block enabling disulfide crosslinking of the formed micelle core to stabilize its structure regardless of temperature and dilution. The cloud points of the PDEAm block and the diblock copolymer were investigated by turbidimetry and fluorescence spectroscopy. The temperature-dependent formation of micelles was analyzed by dynamic light scattering (DLS) and elucidated in detail by an analytical ultracentrifuge (AUC), which provided detailed insights into the solution dynamics between polymers and assembled micelles as a function of temperature. Finally, the micelles were investigated for their applicability as gene delivery vectors by evaluation of cytotoxicity, pDNA binding, and transfection efficiency using HEK293T cells. The investigations showed that core-crosslinking resulted in a 13-fold increase in observed transfection efficiency. Our study presents a comprehensive investigation from polymer synthesis to an in-depth physicochemical characterization and biological application of a crosslinked micelle system including stimuli-responsive behavior.

Correlation between Protonation of Tailor-Made Polypiperazines and Endosomal Escape for Cytosolic Protein Delivery.

Responsive polymers, which become protonated at decreasing pH, are considered a milestone in the development of synthetic cell entry vectors. Exact correlations between their properties and their ability to escape the endosome, however, often remain elusive due to hydrophobic interactions or limitations in the design of water-soluble materials with suitable basicity. Here, we present a series of well-defined, hydrophilic polypiperazines, where systematic variation of the amino moiety facilitates an unprecedented fine-tuning of the basicity or pKa value within the physiologically relevant range (pH 6-7.4). Coincubation of HEK 293T cells with various probes, including small fluorophores or functioning proteins, revealed a rapid increase of endosomal release for polymers with pKa values above 6.5 or 7 in serum-free or serum-containing media, respectively. Similarly, cytotoxic effects became severe at increased pKa values (>7). Although the window for effective transport appears narrow, the discovered correlations offer a principal guideline for the design of effective polymers for endosomal escape.

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.

Solely aqueous formulation of hydrophobic cationic polymers for efficient gene delivery.

Cationic polymers are promising gene delivery vectors due to their ability to bind and protect genetic material. The introduction of hydrophobic moieties into cationic polymers can further improve the vector efficiency, but common formulations of hydrophobic polymers involve harsh conditions such as organic solvents, impairing intactness and loading efficiency of the genetic material. In this study, a mild, aqueous formulation method for the encapsulation of high amounts of genetic material is presented. A well-defined pH-responsive hydrophobic copolymer, i.e. poly((n-butylmethacrylate)-co-(methylmethacrylate)-co-(2-(dimethylamino) ethylmethacrylate)), (PBMD) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Exploiting the pH-dependent solubility behavior of the polymer, stable pDNA loaded nanoparticles were prepared and characterized using analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). This novel formulation approach showed high transfection efficiencies in HEK293T cells, while requiring 5- to 10-fold less pDNA compared to linear polyethylenimine (LPEI), in particular at short incubation times and in serum-containing media. Furthermore, the formulation was successfully adopted for siRNA and mRNA encapsulation and the commercially approved polymer Eudragit® E(PO/100). Overall, the aqueous formulation approach, accompanied by a tailor-made hydrophobic polymer and detailed physicochemical and application studies, led to improved gene delivery vectors with high potential for further applications.

Formulation of Liver-Specific PLGA-DY-635 Nanoparticles Loaded with the Protein Kinase C Inhibitor Bisindolylmaleimide I.

Bisindolylmaleimide I (BIM-I) is a competitive pan protein kinase C inhibitor with anti-inflammatory and anti-metastatic properties, suggested to treat inflammatory diseases and various cancer entities. However, despite its therapeutic potential, BIM-I has two major drawbacks, i.e., it has a poor water solubility, and it binds the human ether-à-go-go-related gene (hERG) ion channels, potentially causing deadly arrhythmias. In this case, a targeted delivery of BIM-I is imperative to minimize peripheral side effects. To circumvent these drawbacks BIM-I was encapsulated into nanoparticles prepared from poly(lactic-co-glycolic acid) (PLGA) functionalized by the near-infrared dye DY-635. DY-635 served as an active targeting moiety since it selectively binds the OATP1B1 and OATP1B3 transporters that are highly expressed in liver and cancer cells. PLGA-DY-635 (BIM-I) nanoparticles were produced by nanoprecipitation and characterized using dynamic light scattering, analytical ultracentrifugation, and cryogenic transmission electron microscopy. Particle sizes were found to be in the range of 20 to 70 nm, while a difference in sizes between the drug-loaded and unloaded particles was observed by all analytical techniques. In vitro studies demonstrated that PLGA-DY-635 (BIM-I) NPs prevent the PKC activation efficiently, proving the efficacy of the inhibitor after its encapsulation, and suggesting that BIM-I is released from the PLGA-NPs. Ultimately, our results present a feasible formulation strategy that improved the cytotoxicity profile of BIM-I and showed a high cellular uptake in the liver as demonstrated in vivo by intravital microscopy investigations.