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.

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 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.

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.

Physicochemical and biological evaluation of siRNA polyplexes based on PEGylated Poly(amido amine)s.

PURPOSE: Use of RNA interference as novel therapeutic strategy is hampered by inefficient delivery of its mediator, siRNA, to target cells. Cationic polymers have been thoroughly investigated for this purpose but often display unfavorable characteristics for systemic administration, such as interactions with serum and/or toxicity. METHODS: We report the synthesis of a new PEGylated polymer based on biodegradable poly(amido amine)s with disulfide linkages in the backbone. Various amounts of PEGylated polymers were mixed with their unPEGylated counterparts prior to polyplex formation to alter PEG content in the final complex. RESULTS: PEGylation effectively decreased polyplex surface charge, salt- or serum-induced aggregation and interaction with erythrocytes. Increasing amount of PEG in formulation also reduced its stability against heparin displacement, cellular uptake and subsequent silencing efficiency. Yet, for polyplexes with high PEG content, significant gene silencing efficacy was found, which was combined with almost no toxicity. CONCLUSIONS: PEGylated poly(amido amine)s are promising carriers for systemic siRNA delivery in vivo.

Disulfide-based poly(amido amine)s for siRNA delivery: effects of structure on siRNA complexation, cellular uptake, gene silencing and toxicity.

PURPOSE: RNA interference (RNAi) is a process by which small interfering RNAs (siRNA) induce sequence-specific gene silencing. Therefore, siRNA is an emerging promise as a novel therapeutic. In order to realize the high expectations for therapeutic applications, efficient delivery systems for siRNA are necessary. METHODS: In this study, a new series of biodegradable poly(amido amine)s with disulfide linkages in the backbone was synthesized out of N,N'-cystaminebisacrylamide (CBA), 4-amino-1-butanol (ABOL) and ethylene diamine (EDA). Effects of different percentages of butanolic side chains and protonatable fragments in the main chain on siRNA complexation, cellular uptake, gene silencing and toxicity were investigated. RESULTS: Incorporation of EDA in the polymer resulted in increased siRNA condensation. Efficient siRNA condensation was shown to be necessary for cellular uptake; however, excess of polymer decreased siRNA uptake for polymers with high amounts of EDA. Silencing efficiency did not correlate with uptake, since silencing increased with increasing w/w ratio for all polymers. More than 80% knockdown was found for polyplexes formed with polymers containing 25% or 50% EDA, which was combined with low cytotoxicity. CONCLUSIONS: Poly(amido amine)s with minor fractions of protonatable fragments in the main chain are promising carriers for delivery of siRNA.

Improved synthesis strategy of poly(amidoamine)s for biomedical applications: catalysis by "green" biocompatible earth alkaline metal salts.

Poly(amidoamine)s (PAAs) have received significant attention due to their good biocompatibility and fast biodegradation profile which gives these polymers high potential in biomedical applications. Conventional synthesis of PAAs via aza-type Michael addition reaction of primary amines to bis-acrylamides often proceeds slowly and takes several days, which does not allow fast and extensive screening of PAA libraries for their bioactivity. Current investigation was dedicated to the development of catalytic synthesis procedures in order to decrease the polymerization times. The salts of several transition metals, as well as earth alkali metals were studied for their catalytic activity in the polymerization reaction. It was found that the salts of earth alkali metals showed the highest potential in the catalysis of polymerization, whereas the salts of transition metals showed either no effect or even resulted in slowing down the reaction. In particular, the addition of CaCl(2) to the reaction mixtures resulted in remarkable increase of the reaction rate as compared to the system without catalyst. PAAs synthesized by the conventional procedure and those obtained by using CaCl(2) as a catalyst showed no difference in physico-chemical properties as well as in biological activity. The novel synthetic method for PAAs, using catalysts based on earth alkali metals, represents an attractive alternative to currently applied methods. Characteristics of earth alkali metals such as low toxicity and good biocompatibility make them especially useful in the preparation of these polymers for biomedical applications.

Mixtures of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)-g-poly(ethylene glycol) for improved gene delivery.

Branched disulfide-containing poly(amido ethyleneimines) (SS-PAEIs) are biodegradable polymeric gene carrier analogues of the well-studied, nondegradable, and often toxic branched polyethylenimines (bPEIs), but with distinct advantages for cellular transgene delivery. Clinical success of polycationic gene carriers is hampered by obscure design and formulation requirements. This present work reports synthetic and formulation properties for a graft copolymer of poly(ethylene glycol) (PEG) and a branched SS-PAEI, poly(triethylentetramine/cystaminebisacrylamide) (p(TETA/CBA)). Several laboratories have previously demonstrated the advantages of PEG conjugation to gene carriers, but have also shown that PEG conjugation may perturb plasmid DNA (pDNA) condensation, thereby interfering with nanoparticle formation. With this foundation, our studies sought to mix various amounts of p(TETA/CBA) and p(TETA/CBA)-g-PEG2k to alter the relative amount of PEG in each formulation used for polyplex formation. The influence of different PEG/polycation amounts in the formulations on polymer/nucleic acid nanoparticle (polyplex) size, surface charge, morphology, serum stability and transgene delivery was studied. Polyplex formulations were prepared using p(TETA/CBA)-g-PEG2k, p(TETA/CBA), and mixtures of the two species at 10/90 and 50/50 volumetric mixture ratios (wt/wt %), respectively. As expected, increasing the amount of PEG in the formulation adversely affects polyplex formation. However, optimal polymer mixtures could be identified using this facile approach to further clarify design and formulation requirements necessary to understand and optimize carrier stability and biological activity. This work demonstrates the feasibility to easily overcome typical problems observed when polycations are modified and thus avoids the need to synthesize multiple copolymers to identify optimal gene carrier candidates. This approach may be applied to other polycation-PEG preparations to alter polyplex characteristics for optimal stability and biological activity.

Dual bio-responsive gene delivery via reducible poly(amido amine) and survivin-inducible plasmid DNA.

A bioreducible poly(amido amine) (SS-PAA) gene carrier, known as poly (amido-butanol) (pABOL), was used to transfect a variety of cancer and non-cancer cell lines. To obtain cancer-specific transgene expression for therapeutic efficiency in cancer treatment, we constructed survivin-inducible plasmid DNA expressing the soluble VEGF receptor, sFlt-1, downstream of the survivin promoter (pSUR-sFlt-1). Cancer-specific expression of sFlt-1 was observed in the mouse renal carcinoma (RENCA) cell line. pABOL enhanced the efficiency of gene delivery compared to traditional carriers used in the past. Thus, a dual bio-responsive gene delivery system was developed by using bioreducible p(ABOL) for enhanced intracellular gene delivery and survivin-inducible gene expression system (pSUR-sFlt-1 or pSUR-Luc reporter gene) that demonstrates increased gene expression in cancer that has advantages over current gene delivery systems.

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.

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.

Reducible poly(amido ethylenimine) directed to enhance RNA interference.

Designing synthetic macromolecular vehicles with high transfection efficiency and low cytotoxicity has been a major interest in the development of non-viral gene carriers. A reducible poly(amido ethylenimine) (SS-PAEI) synthesized by addition copolymerization of triethylenetetramine and cystamine bis-acrylamide (poly(TETA/CBA)) was used as a carrier for small interference RNA (siRNA). Poly(TETA/CBA) could efficiently condense siRNA to form stable complexes under physiological conditions and perform complete release of siRNA in a reductive environment. When formulated with VEGF-directed siRNA, poly(TETA/CBA) demonstrated significantly higher suppression of VEGF than linear-polyethylenimine (PEI) (L-PEI, 25kDa) in human prostate cancer cells (PC-3). After 5h of transfection, substantial dissociation and intracellular distribution of siRNA was observed in the poly(TETA/CBA) formulation, but not in the L-PEI formulation. The triggered release of siRNA by reductive degradation of poly(TETA/CBA) in the cytoplasm may affect the RNAi activity by increasing cytoplasmic availability of siRNA. These results suggest that the rational design of non-viral carriers should involve considerations for intracellular dissociation and trafficking of a nucleic acid drug to maximize its effect, in conjunction with formation of stable complexes under physiological conditions.

Linear poly(amido amine)s with secondary and tertiary amino groups and variable amounts of disulfide linkages: synthesis and in vitro gene transfer properties.

A group of novel poly(amido amine) homo- and copolymers (PAAs) containing secondary and tertiary amine groups in their main chain and different structures in the bisacrylamide segments were synthesized and evaluated as non-viral gene delivery vectors. Among these, also the disulfide-containing cystaminebisacrylamide was employed as a (co)monomer, yielding PAAs with variable amounts of bioreducible disulfide linkages in the main chain. Michael addition the trifunctional 1-(2-aminoethyl) piperazine to equimolar amounts of the appropriate bis(acrylamide) yielded linear polymers as was elucidated by their (13)C NMR spectra. The polymers possess buffering capacities between pH 5.1 and pH 7.4 higher than branched polyethylenimine (pEI) and are able to efficiently condense DNA into nanosized (<150 nm) and positively charged complexes. Transfection experiments with COS-7 cells showed that polyplexes from PAAs with disulfide linkages give significant higher transfections than those from PAAs lacking the disulfide linkage, and XTT assays showed that these polymers are essentially non-toxic. Variation of the disulfide content revealed that polyplexes of PAA copolymers with appropriate disulfide content have largely improved biophysical properties, yielding enhanced levels of gene expression along with low toxicity. The results demonstrate that bioreducible poly(amido amine)s are a very promising class of polymers for safe and efficient gene delivery.

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.

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.

A versatile family of degradable non-viral gene carriers based on hyperbranched poly(ester amine)s.

A variety of degradable hyperbranched poly(ester amine)s containing primary, secondary and tertiary amino groups, were synthesized and evaluated as non-viral gene carriers. The polymers were obtained in high yields through a Michael-type conjugate addition of diacrylate monomers with trifunctional amine monomers. Analysis of degradation products using liquid chromatography-mass spectroscopy (LC-MS) demonstrated that all poly(ester amine)s had a hyperbranched structure with a degree of branching of approximately 0.30. These poly(ester amine)s were readily water-soluble and degradable under physiological conditions (pH 7.4, 37 degrees C), in which more than 10% ester bonds were hydrolyzed within 4 h. Moreover, these hyperbranched poly(ester amine)s showed high buffering capacities between pH 5.1 and 7.4. Three out of nine synthesized polymers, i.e. p(HDDA-AEP), p(HDDA-AMP), and p(BDDA-AMP), were shown to effectively condense plasmid DNA into small-sized (approximately 94-135 nm) and positively charged complexes. Polymer/DNA complexes ('polyplexes') based on these three polymers, and larger complexes of p(BDDA-AEP) (approximately 497 nm) were able to transfect COS-7 cells in vitro. Importantly, the transfection activity of polyplexes was preserved in the presence of serum proteins. The highest transfection level was observed for p(HDDA-AEP) polyplex which had a transfection efficiency higher than or comparable to that polyplexes of polyethylenimine (PEI) and poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA). Furthermore, these poly(ester amine)s revealed no or low cytotoxicity. These results demonstrated that hyperbranched poly(ester amine)s can be applied as safe and efficient gene delivery polymers.

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.

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.