A poly(amidoamine)-based polymeric nanoparticle platform for efficient in vivo delivery of mRNA.

The successful use of mRNA vaccines enabled and accelerated the development of several new vaccine candidates and therapeutics based on the delivery of mRNA. In this study, we developed bioreducible poly(amidoamine)-based polymeric nanoparticles (PAA PNPs) for the delivery of mRNA with improved transfection efficiency. The polymers were functionalized with chloroquinoline (Q) moieties for improved endosomal escape and further stabilization of the mRNA-polymer construct. Moreover, these PAAQ polymers were covalently assembled around a core of multi-armed ethylenediamine (Mw 800, 2 % w/w) to form a pre-organized polymeric scaffolded PAAQ (ps-PAAQ) as a precursor for the formation of the mRNA-loaded nanoparticles. Transfection of mammalian cell lines with EGFP mRNA loaded into these PNPs showed a favorable effect of the Q incorporation on GFP protein expression. Additionally, these ps-PAAQ NPs were co-formulated with PEG-polymer coatings to shield the positive surface charge for increased stability and better in vivo applicability. The ps-PAAQ NPs coated with PEG-polymer displayed smaller particle size, electroneutral surface charge, and higher thermal stability. Importantly, these nanoparticles with both Q and PEG-polymer coating induced significantly higher luciferase activity in mice muscle than uncoated ps-PAAQ NPs, following intramuscular injection of PNPs loaded with luciferase mRNA. The developed technology is broadly applicable and holds promise for the development of new nucleotide-based vaccines and therapeutics in a range of infectious and chronic diseases.

Responsive crosslinked polymer nanogels for imaging and therapeutics delivery.

Water-soluble, nano-sized crosslinked polymer networks, or nanogels, are delivery vehicles, which have highly interesting properties for therapeutic delivery and imaging. Nanogels may also possess responsive properties, depending on the employed polymers, allowing controlled release of therapeutics or image contrast generation upon exposure to physical or (bio)chemical cues. In this review, polymer nanogels are explored for application in imaging as well as for controlled drug and gene delivery. Moreover, nanogels are explored as responsive biomaterials and future applications are highlighted.

Polymers and hydrogels for local nucleic acid delivery.

The potential of gene therapy for the treatment for chronic and life-threatening diseases has been seen for a long time, but widespread applications are still hampered by the difficulties to deliver the highly charged and large nucleic acid molecules to their intracellular targets. More recently, investigators have been aiming for local delivery of nucleic acids mostly by the use of hydrogels. In this way, in vivo efficacy can be enhanced by avoiding the target transport challenges and at the same time limit off-target effects. In these systems, nucleic acids are entrapped within hydrogels, either as conjugates or as polyplex particles, for local and controlled release. There are numerous design features in the selection of polymers, for both particle and hydrogel formation that should be considered to achieve efficient local nucleic acid delivery. Therefore, this review focusses on the rational design of polymeric and hydrogel materials for local gene therapy applications.

Modular Synthesis of Bioreducible Gene Vectors through Polyaddition of N,N'-Dimethylcystamine and Diglycidyl Ethers.

Bioreducible, cationic linear poly(amino ether)s (PAEs) were designed as promising gene vectors. These polymers were synthesized by the reaction of a disulfide-functional monomer, N,N'-dimethylcystamine (DMC), and several different diglycidyl ethers. The resulting PAEs displayed a substantial buffer capacity (up to 64%) in the endosomal acidification region of pH 7.4⁻5.1. The PAEs condense plasmid DNA into 80⁻200 nm sized polyplexes, and have surface charges ranging from +20 to +40 mV. The polyplexes readily release DNA upon exposure to reducing conditions (2.5 mM DTT) due to the cleavage of the disulfide groups that is present in the main chain of the polymers, as was demonstrated by agarose gel electrophoresis. Upon exposing COS-7 cells to polyplexes that were prepared at polymer/DNA w/w ratios below 48, cell viabilities between 80⁻100% were observed, even under serum-free conditions. These polyplexes show comparable or higher transfection efficiencies (up to 38%) compared to 25 kDa branched polyethylenimine (PEI) polyplexes (12% under serum-free conditions). Moreover, the PAE-based polyplexes yield transfection efficiencies as high as 32% in serum-containing medium, which makes these polymers interesting for gene delivery applications.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

Disulfide-functional poly(amido amine)s with tunable degradability for gene delivery.

Controlled degradability in response to the local environment is one of the most effective strategies to achieve spatiotemporal release of genes from a polymeric carrier. Exploiting the differences in reduction potential between the extracellular and intracellular environment, disulfides are frequently incorporated into the backbone of polymeric drug delivery agents to ensure efficient intracellular release of the payload. However, although to a lesser extent, reduction of disulfides may also occur in the extracellular environment and should be prevented to avoid premature release. Accurate control over the stability of disulfide linkages enables the optimization of polymeric carriers for efficient drug delivery. Bioreducible poly(amido amine)s (PAAs) with varying degrees of steric hindrance adjacent to the disulfide bonds (0, 2 or 4 methyl groups) were prepared in order to obtain carriers with controlled stability. The degradation behavior of these PAA-polymers was evaluated under different reducing conditions and their in vitro toxicities and transfection efficiencies were assessed. Degradation of the PAA-based polyplexes consistently required higher reducing strengths as the steric hindrance near the disulfide bonds increased. Polyplexes based on 2-methyl cystamine disulfide based PAA polymer (PAA2m) remained stable under extracellular glutathione concentrations (0.001-0.01mM), while degrading within 1h under reducing conditions similar to those in the intracellular environment (1-10mM glutathione). This polymer exhibited excellent transfection capabilities, with efficiencies up to 90% of transfected cells. PAA0m showed slightly reduced transfection properties compared to PAA2m, likely due to premature degradation. The severely hindered PAA4m, however, displayed increased toxicity, accompanied by reduced transfection efficiency, as a result of its exceptional stability. These results demonstrate the feasibility of introducing steric hindrance near the disulfide moiety to tune polyplex stability against bioreduction, and show that PAA2m is a promising polymer to be further developed for gene therapy.

Surfactant-free preparation of highly stable zwitterionic poly(amido amine) nanogels with minimal cytotoxicity.

Narrowly dispersed zwitterionic poly(amido amine) (PAA) nanogels with a diameter of approximately 100nm were prepared by a high-yielding and surfactant-free, inverse nanoprecipitation of PAA polymers. The resulting, negatively charged, nanogels (PAA-NG1) were functionalized with N,N-dimethylethylenediamine via EDC/NHS coupling chemistry. This resulted in nanogels with a positive surface charge (PAA-NG2). Both types of nanogels were fluorescently labelled via isothiocyanate coupling. PAA-NG1 displays high colloidal stability both in PBS and Fetal Bovine Serum solution. Moreover, both nanogels exhibit a distinct zwitterionic swelling profile in response to pH changes. Cellular uptake of FITC-labelled nanogels with RAW 264.7, PC-3 and COS-7 cells was evaluated by fluorescence microscopy. These studies showed that nanogel surface charge greatly influences nanogel-cell interactions. The PAA polymer and PAA-NG1 showed minimal cell toxicity as was evaluated by MTT assays. The findings reported here demonstrate that PAA nanogels possess interesting properties for future studies in both drug delivery and imaging. STATEMENT OF SIGNIFICANCE: The use of polymeric nanoparticles in biomedical applications such as drug delivery and imaging, shows great potential for medical applications. However, these nanoparticles are often not stable in biological environments. Zwitterionic polymers have shown excellent biocompatibility, but these materials are not easily degradable in biological environments. With the aim of developing a nanoparticle for drug delivery and imaging we synthesized a biomimetic and readily biodegradable zwitterionic polymer, which was incorporated into nanogels. These nanogels showed excellent stability in the presence of serum and minimal cytotoxicity, which was tested in three cell lines. Because of their negative surface charge and excellent serum stability, these nanogels are therefore promising carriers for drug delivery and molecular imaging.

Poly(Amido Amine)s Containing Agmatine and Butanol Side Chains as Efficient Gene Carriers.

A new type of bioreducible poly(amido amine) copolymer is synthesized by the Michael addition polymerization of cystamine bisacrylamide (CBA) with 4-aminobutylguanidine (agmatine, AGM) and 4-aminobutanol (ABOL). Since the positively charged guanidinium groups of AGM and the hydroxybutyl groups of ABOL in the side chains have shown to improve the overall transfection efficiency of poly(amido amine)s, it is hypothesized that poly(CBA-ABOL/AGM) synthesized at the optimal ratio of both components would result in high transfection efficiency and minimal toxicity. In this study, a series of the poly(CBA-ABOL/AGM) copolymers is synthesized as gene carriers. The polymers are characterized and luciferase transfection efficiencies of the polymers in various cell lines are investigated to select the ideal ratio between AGM and ABOL. The poly(CBA-ABOL/AGM) containing 80% AGM and 20% ABOL has shown the best transfection efficiency with the lowest cytotoxicity, indicating that this polymer is very promising as a potent and nontoxic gene carrier.

Multilayered Thin Films from Boronic Acid-Functional Poly(amido amine)s As Drug-Releasing Surfaces.

PURPOSE: To evaluate the potential of poly(amido amine)-based multilayered thin films in surface mediated drug release. METHODS: Multilayered thin films were prepared from copolymers of phenylboronic acid-functional poly(amido amine)s and chondroitin sulfate (ChS) in the presence of Alizarin Red S (ARS) as a reporter molecule. Multilayer buildup and ARS incorporation were evaluated with UV-vis spectroscopy. Glucose responsiveness of the multilayers was investigated. Finally, cellular uptake of ARS by COS-7 cells grown on the films was assessed. RESULTS: Multilayers based on alcohol containing polymers (ABOL-BA-PAA#ChS + ARS) displayed higher ARS incorporation than multilayers based on amine-containing polymers (DAB-BA-PAA#ChS + ARS). At physiological pH, a swift initial release of up to ~40% of the ARS content was observed during the first 12 h of incubation, followed by a much slower, gradual release of ARS. The multilayers were further evaluated by culturing COS-7 cells on top of multilayer-coated well plates. Cellular uptake of the fluorescent ARS-boronate ester was quantified through flow cytometry, and a maximum uptake of up to 30% was observed. Confocal microscopy confirmed the presence of ARS-boronate ester-containing particles in the nuclei of cells. CONCLUSIONS: The investigated multilayered thin films are effective in surface-mediated delivery of the model compound ARS. These multilayered surfaces are promising as drug-releasing delivery surface for coating stents, prostheses, and other implants.

Multilayered thin films from poly(amido amine)s and DNA.

Dip-coated multilayered thin films of poly(amido amine)s (PAAs) and DNA have been developed to provide surfaces with cell-transfecting capabilities. Three types of PAAs, differing in side chain functional groups, were synthesized and characterized for their properties in forming multilayered structures with ultrasonicated calf thymus DNA (CTDNA) as model DNA. All three polymers display a multilayer build-up in linear profiles as demonstrated by UV spectroscopy. More highly charged side chains were found to provide the lowest deposition of DNA. Surface profiles of the obtained films were investigated by atomic force microscopy (AFM) and static water contact angle measurements to reveal complete surface coverage after at least four layer pair depositions, where alternating patterns of surface profiles were observed depending on whether the cationic polymer or the anionic DNA layer was on top. The stability of the formed surfaces was investigated in vitro under physiological and reductive conditions. Owing to the presence of disulfide bonds in the PAA main chain, the films were readily degraded in the presence of 1mM of DTT in vitro. Under non-reductive physiological conditions, two of the thicker films underwent thermodynamic rearrangement, which resulted in release of approximately half of the incorporated material within 1h, which was caused by the physiological salt concentration. Further, this unpacking phenomenon proved useful in transfecting COS-7 cells seeded on top of these multilayers containing functional plasmid DNA encoding for green fluorescence protein (GFP). Two out of the three different multilayers facilitated good COS-7 cell attachment, proliferation, and transfection in vitro within 2d ays of culture. Fluorescence staining further revealed the presence of DNA-containing released film material among cultured cells. The present work demonstrates the possibility of coating surfaces with thin films that are conveniently adjustable in thickness and amount of active agent to provide cell-transfecting functionality. In this manner transfection can be achieved by simply culturing cells on a multilayer-coated surface in their optimal culture condition (in the presence of serum) and without the need of removing the transfection agent to avoid cytotoxicity.

Multilayered Thin Films from Boronic Acid-Functional Poly(amido amine)s.

PURPOSE: To investigate the properties of phenylboronic acid-functional poly(amido amine) polymers (BA-PAA) in forming multilayered thin films with poly(vinyl alcohol) (PVA) and chondroitin sulfate (ChS), and to evaluate their compatibility with COS-7 cells. METHODS: Copolymers of phenylboronic acid-functional poly(amido amine)s, differing in the content of primary amine (DAB-BA-PAA) or alcohol (ABOL-BA-PAA) side groups, were synthesized and applied in the formation of multilayers with PVA and ChS. Biocompatibility of the resulting films was evaluated through cell culture experiments with COS-7 cells grown on the films. RESULTS: PVA-based multilayers were thin, reaching ~100 nm at 10 bilayers, whereas ChS-based multilayers were thick, reaching ~600 nm at the same number of bilayers. All of the multilayers are stable under physiological conditions in vitro and are responsive to reducing agents, owing to the presence of disulfide bonds in the polymers. PVA-based films were demonstrated to be responsive to glucose at physiological pH at the investigated glucose concentrations (10-100 mM). The multilayered films displayed biocompatibility in cell culture experiments, promoting attachment and proliferation of COS-7 cells. CONCLUSIONS: Responsive thin films based on boronic acid functional poly(amido amine)s are promising biocompatible materials for biomedical applications, such as drug releasing surfaces on stents or implants. Graphical Abstract Layer-by-Layer Assembly.

Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection.

Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D. Therefore, COS-7 cells were cultured on top of multilayer films formed by layer-by-layer dipcoating of these polymers with GFP-encoded pDNA, and the effect of the number of layers and cell seeding density on the transfection efficiency was evaluated. Multilayer films with pCABOL were found to be superior to pCHIS in facilitating transfection, which was attributed to higher incorporation of pDNA and release of the transfection agent. High amounts of transfected cells were obtained on pCABOL films, correlating proportionally over a wide range with seeding density. Optimal transfection efficiency was obtained with pCABOL films composed of 10 bilayers. Further increase in the number of bilayers only marginally increased transfection efficiency. Using the optimal multilayer and cell seeding conditions, pCABOL multilayers were fabricated on poly(ε-caprolactone) (PCL), heparinized PCL (PCL-HEP), and poly(lactic acid) (PLA) disks as examples of common biomedical supports. The multilayers were found to completely mask the properties of the original substrates, with significant improvement in cell adhesion, which is especially pronounced for PCL and PLA disks. With all these substrates, transfection efficiency was found to be in the range of 25-50% transfected cells. The pCABOL/pDNA multilayer films can also conveniently add transfection capability to 3D scaffolds. Significant improvement in cell adhesion was observed after multilayer coating of 3D-plotted fibers of PCL (with and without an additional covalent heparin layer), especially for the PCL scaffold without heparin layer and transfection was observed on both 3D PCL and PCL-HEP scaffolds. These results show that layer-by-layer dip-coating of pCABOL with functional DNA is an easy and inexpensive method to introduce transfection capability to biomaterials of any nature and shape, which can be beneficially used in various biomedical and tissue engineering applications.

Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy.

Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated polyplexes had a Z-averaged diameter of 108nm and a zeta potential of +29mV. We evaluated polyplexes coated with HA of different molecular weights (22kDa, 137kDa and 2700kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated polyplexes had good mobility in bovine vitreous humor, similar to polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700kDa). However, contrary to PEGylated polyplexes, HA-coated polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular weight HA (22kDa) was shown to induce the highest expression. Taken together our experiments show that HA-coating of nonviral gene complexes is an interesting approach towards retinal gene therapy by intravitreal administration. To our knowledge, this is the first time electrostatic HA-coating of polyplexes with different molecular weights has been evaluated in terms of their suitability for intravitreal delivery of therapeutic nucleic acids towards the retina.

Microfluidic preparation of polymer-nucleic acid nanocomplexes improves nonviral gene transfer.

As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

Development and in vitro evaluation of antigen-loaded poly(amidoamine) nanoparticles for respiratory epithelium applications.

A poly(amidoamine) with disulfide linkages in the main chain and 4-hydroxybutyl and ω-carboxy-PEG groups (9:1 ratio) as side chains was prepared by Michael addition polymerization of cystamine bisacrylamide with 4-hydroxybutylamine and ω-carboxy-PEG-amine. To develop therapeutic protein formulations for improved delivery of antigen via the intranasal route, nanoparticles were prepared from this polymer by self-assembly with p24 or ovalbumin as the model proteins and CpG as the adjuvant. The nanoparticles incorporated the antigens and adjuvant from the feed solution with high efficiency (∼90 %) and have sizes of 112 and 169 nm, respectively, with low positive surface charge (∼+2 mV). Formulations of the nanoparticles were shown to be nontoxic and stable for at least 10 days at room temperature. Their capacity to pass through epithelial and endothelial cell layers was evaluated in vitro by using a respiratory mucosa-like barrier model in which monolayers of NCI H441 respiratory epithelial cells and ISO-HAS-1 endothelial cells were co-cultured on both sides of a transwell filter membrane. It was shown that p24 incorporated in the nanoparticles was transported with >140 % greater efficiency through the two contact-inhibited layers than p24 in its free form, whereas incorporation of ovalbumin in the nanoparticles leads to a 40 % decrease in transport efficiency relative to the free antigen.

Design and physicochemical characterization of poly(amidoamine) nanoparticles and the toxicological evaluation in human endothelial cells: applications to peptide delivery to the brain.

In this study, we investigated nanoparticles formulated by self-assembly of a biodegradable poly(amidoamine) (PAA) and a fluorescently labeled peptide, in their capacity to internalize in endothelial cells and deliver the peptide, with possible applications for brain drug delivery. The nanoparticles were characterized in terms of size, surface charge, and loading efficiency, and were applied on human cerebral microvascular endothelial cells (hCMEC/D3) and human umbilical vein endothelial cells (Huvec) cells. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially nontoxic, and the peptide was successfully internalized into cells. The results indicate that these PAAs have an excellent property as nontoxic carriers for intracellular protein and peptide delivery, and provide opportunities for novel applications in the delivery of peptides to endothelial cells of the brain.

Carbohydrate-interactive pDNA and siRNA gene vectors based on boronic acid functionalized poly(amido amine)s.

In order to evaluate the influence of incorporation of boronic acid groups on the properties of poly(amido amine)s as gene vectors, a novel poly(amido amine) copolymer p(CBA-ABOL/2AMPBA) containing ortho-aminomethylphenylboronic acid (2AMPBA) moieties was prepared by Michael-type polyaddition of a mixture of 1,4-aminobutanol (ABOL) and 2-((4-aminobutylamino)methyl)phenyl boronic acid to N,N'-cystamine bisacrylamide (CBA). It appeared that the presence of the boronic acid moieties as side groups along the polymer chain strongly enhances the stability of the self-assembled nanoparticles and nanosized polyplexes formed from this polymer; no aggregation was observed after storage for 6days at 37°C. This strong stabilization can be attributed to intermolecular Lewis acid-base interactions between the 2AMPBA groups and the alcohol and amine groups present in the polymer, leading to dynamical (reversible) crosslinking in the nanoparticles. Moreover, since the boronic acids can reversibly form boronic esters with vicinal diol groups, the presence of the 2AMPBA groups add carbohydrate-interactive properties to these polymers that strongly influence their behavior as gene delivery vectors. DNA transfection with p(CBA-ABOL/2AMPBA) polyplexes gave transfection efficiencies that were approximately similar to commercial PEI in different cell lines (COS-7, HUH-6 and H1299-Fluc), but lower than those obtained with reference polyplexes from p(CBA-ABOL). It is hypothesized that the uptake of the boronated polyplexes is suppressed by binding to the glycocalyx of the cells. This is supported by the observation that addition of sorbitol or dextran to the transfection medium significantly enhances the transfection efficiency, which can be attributed to increased cellular uptake of the polyplexes due to boronic ester formation with these agents. AFM, SEM and confocal microscopy showed that polyplexes of p(CBA-ABOL/2AMPBA) become decorated with a dextran layer in the presence of 0.9% (w/v) dextran in the polyplex medium. In siRNA-mediated gene silencing experiments the use of p(CBA-ABOL/2AMPBA) as polymeric vector gave a knockdown of luciferase expression in H1229-Fluc cells of 35%. Also in this case addition of 0.9% (w/v) sorbitol or dextran to the transfection medium strongly increased the knockdown efficiency to 59% and 76%, respectively.

Bioresponsive poly(amidoamine)s designed for intracellular protein delivery.

Poly(amidoamine)s with bioreducible disulfide linkages in the main chain (SS-PAAs) and pH-responsive, negatively charged citraconate groups in the sidechain have been designed for effective intracellular delivery and release of proteins with a net positive charge at neutral pH. Using lysozyme as a cationic model protein these water soluble polymers efficiently self-assemble into nanocomplexes by charge attraction. At pH5 (the endosomal pH) the amide linkages connecting the citraconate groups in the sidechains of the SS-PAAs are hydrolyzed by intramolecular catalysis, resulting in expulsion of the negative citraconate groups and formation of protonated amine groups, resulting in charge reversal of the polymeric carrier from negative to positive. The concomitant endosomal buffering effect and increased polymer-endosomal membrane interactions are considered to lead to increased protein delivery into the cytosol. Besides destabilization of the polymer-protein nanoparticles by the charge reversal effect, intracellular cleavage of disulfide linkages in the polymer ensure further unpacking of the protein in the cytosol. Cellinternalization and cytotoxicity experiments with primary human umbilical vein endothelial cells (HUVEC) showed that the SS-PAA-based nanocomplexes were essentially non-toxic, and that lysozyme is successfully internalized into HUVEC. The results indicate that these charge reversal SS-PAAs have excellent properties as non-toxic intracellular delivery systems for cationic proteins.