Chlorophyllin-Containing Copolymers and Their Responsive Properties.

Copper-chlorophyllin is a water-soluble derivative of chlorophylls and shows low cytotoxicity and antimutagenic properties in cultured cells. It has multiple applications, including its use as a photosensitizer in photothermal therapy because of its green light-activated photothermal performance. In this work, it was copolymerized with a poly(ethylene glycol) methacrylic monomer to yield random copolymers by free radical polymerization, which showed dual temperature- and pH-dependent phase transitions in aqueous solutions. The cloud points of the copolymer solutions were raised by lowering the pH of the aqueous solutions due to the protonation of the carboxylic groups on the chlorophyllin moieties, which decreased the overall hydrophilicity of the polymers. At low pH values, complete protonation of the carboxylic acid groups of the chlorophyllin moieties led to an irreversible aggregation of the copolymers in water. The incorporation of chlorophyllin in the copolymer improved its stability over its single molecular form.

Injectable Lyophilized Chitosan-Thrombin-Platelet-Rich Plasma (CS-FIIa-PRP) Implant to Promote Tissue Regeneration: In Vitro and Ex Vivo Solidification Properties.

Freeze-dried chitosan formulations solubilized in platelet-rich plasma (PRP) are currently evaluated as injectable implants with the potential for augmenting the standard of care for tissue repair in different orthopedic conditions. The present study aimed to shorten the solidification time of such implants, leading to an easier application and a facilitated solidification in a wet environment, which were direct demands from orthopedic surgeons. The addition of thrombin to the formulation before lyophilization was explored. The challenge was to find a formulation that coagulated fast enough to be applied in a wet environment but not too fast, which would make handling/injection difficult. Four thrombin concentrations were analyzed (0.0, 0.25, 0.5, and 1.0 NIH/mL) in vitro (using thromboelastography, rheology, indentation, syringe injectability, and thrombin activity tests) as well as ex vivo (by assessing the implant's adherence to tendon tissue in a wet environment). The biomaterial containing 0.5 NIH/mL of thrombin significantly increased the coagulation speed while being easy to handle up to 6 min after solubilization. Furthermore, the adherence of the biomaterial to tendon tissues was impacted by the biomaterial-tendon contact duration and increased faster when thrombin was present. These results suggest that our biomaterial has great potential for use in regenerative medicine applications.

Adjuvant lipidoid-substituted lipid nanoparticles augment the immunogenicity of SARS-CoV-2 mRNA vaccines.

Lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccineare a promising platform to prevent infectious diseases as demonstrated by the recent success of SARS-CoV-2 mRNA vaccines. To avoid immune recognition and uncontrolled inflammation, nucleoside-modified mRNA is used. However, such modification largely abrogates the innate immune responses that are critical to orchestrating robust adaptive immunity. Here we develop an LNP component-an adjuvant lipidoid-that can enhance the adjuvanticity of mRNA-LNP vaccines. Our results show that partial substitution of ionizable lipidoid with adjuvant lipidoid not only enhanced mRNA delivery, but also endowed LNPs with Toll-like receptor 7/8-agonistic activity, which significantly increased the innate immunity of the SARS-CoV-2 mRNA-LNP vaccine with good tolerability in mice. Our optimized vaccine elicits potent neutralizing antibodies against multiple SARS-CoV-2 pseudovirus variants, strong Th1-biased cellular immunity, and robust B cell and long-lived plasma cell responses. Importantly, this adjuvant lipidoid substitution strategy works successfully in a clinically relevant mRNA-LNP vaccine, demonstrating its translational potential.

Purification and Surface Modification of Chitosan-based Polyplexes Using Tangential Flow Filtration and Coating by Hyaluronic Acid.

Chitosan (CS)-based polyplexes are produced by spontaneous electrostatic association with nucleic acids using CS in excess. Interactions of positively charged polyplexes, and the unbound CS, with negatively charged blood components limit the applicable dosage of such polymeric nanoparticles (NPs) and development of formulations with improved hemocompatibility and transfection efficiency is needed. Here, we introduce a strategy based on Tangential Flow Filtration (TFF) to remove unbound CS, concentrate polyplexes and subsequently coat with hyaluronic acid (HA) to improve hemocompatibility and bioactivity. Optimal TFF conditions were established. A library of HA with different molecular weights and degrees of sulfation was used at different carboxyl + sulfate to phosphate ratios for polyplex coating, bioactivity and hemocompatibility assessment. A systematic optimization of TFF conditions allowed for purification of polylpexes from excess unbound CS and subsequent coating with HA. Except for high molecular weight HA, for which macroscopic aggregation was observed, both sulfated and non-sulfated HAs resulted in small sized and homogenous coated complexes. However, sulfated HAs displayed higher stability during the second filtration process indicating their stronger binding affinity to polyplexes. Finally, we found that low molecular weight HA-coated polyplexes have equivalent silencing efficiency in vitro and improved hemocompatibility compared to uncoated polyplexes.

Chitosan-platelet-rich plasma implants improve rotator cuff repair in a large animal model: Pilot study.

Freeze-dried formulations of chitosan can be solubilized in platelet-rich plasma (PRP) to form injectable implants that are used as an adjunct treatment during surgical repair of the rotator cuff. The purpose of the current study was to assess chitosan-PRP implant residency, test safety, and assess efficacy over standard-of-care controls in a sheep model of rotator cuff repair. The infraspinatus tendon was transected unilaterally and immediately repaired with suture anchors in 22 skeletally mature ewes. In treatment groups, formulations containing chitosan, trehalose, and calcium chloride were solubilized with autologous leukocyte-rich PRP and injected at the tendon-bone interface and on top of the repaired site (1 mL or 2 mL doses). Implant residency was assessed histologically at 1 day. Outcome measures included MRI assessment at baseline, 6 weeks, and 12 weeks, histopathology and clinical pathology. Chitosan-PRP implants were resident at the injection site at 1 day and induced recruitment of polymorphonuclear cells. The tendon gap, which corresponds to the length of abnormally hyperintense tissue attached to the humeral head, was decreased by treatment with the 2 mL dose when compared to controls at 12 weeks on MRI images. Some histological features were improved by the 2 mL dose treatment compared to controls at 12 weeks. There was no treatment-specific effect on all standard safety outcome measures, which suggests high safety. This study provides preliminary evidence on the safety and efficacy of chitosan-PRP implants in a large animal model that could potentially be translated to a clinical setting.

Vaccine Technologies and Platforms for Infectious Diseases: Current Progress, Challenges, and Opportunities.

Vaccination is a key component of public health policy with demonstrated cost-effective benefits in protecting both human and animal populations. Vaccines can be manufactured under multiple forms including, inactivated (killed), toxoid, live attenuated, Virus-like Particles, synthetic peptide, polysaccharide, polysaccharide conjugate (glycoconjugate), viral vectored (vector-based), nucleic acids (DNA and mRNA) and bacterial vector/synthetic antigen presenting cells. Several processes are used in the manufacturing of vaccines and recent developments in medical/biomedical engineering, biology, immunology, and vaccinology have led to the emergence of innovative nucleic acid vaccines, a novel category added to conventional and subunit vaccines. In this review, we have summarized recent advances in vaccine technologies and platforms focusing on their mechanisms of action, advantages, and possible drawbacks.

Chitosan-Platelet-Rich Plasma Implants Improve Rotator Cuff Repair in a Large Animal Model: Pivotal Study.

The purpose of this study was to assess the safety and efficacy of chitosan-platelet-rich plasma (PRP) hybrid implants used as an adjunct to surgical rotator cuff repair in a pivotal Good Laboratory Practice (GLP)-compliant study. The infraspinatus tendon was transected in 48 skeletally mature ewes and repaired with a transosseous-equivalent (TOE) technique. In the two treatment groups, a chitosan-PRP solution was injected at the footprint between the tendon and the bone and on top of the repaired site (2 mL or 3 mL doses, n = 12 per group). To further assess chitosan safety, a chitosan-water solution was injected at the same sites (3 mL, n = 12). Outcome measures included Magnetic Resonance Imaging (MRI) assessment and clinical pathology at 3 months and 6 months and histopathology at 6 months. The tendon gap was decreased at 3 months on MRI images and certain histopathological features were improved at 6 months by chitosan-PRP treatment compared to controls. The group treated with chitosan-water was not different from controls. Chitosan-PRP treatment induced no negative effects in the sheep, which suggests high safety. This study provides further evidence on the safety and efficacy of chitosan-PRP for rotator cuff repair augmentation, which could eventually be used in a clinical setting.

Robust Segmentation-Free Algorithm for Homogeneity Quantification in Images.

OBJECTIVE: Homogeneity is a notion used to describe images in various fields and is often linked to critical aspects of those fields. However, this term is rarely defined in the literature and no gold standard exists for its quantification. A few quantification algorithms have been proposed, but they lack both simplicity and robustness. As a result, the scientific community uses the notion of homogeneity in subjective analysis, preventing objective comparison of a large number of data or of different studies. The main objectives of this manuscript are to propose a definition of homogeneity and an algorithm for its quantification. METHOD: This algorithm, called MASQH, rely on a multi-scale, statistical and segmentation-free approach and outputs a single homogeneity index, which makes it robust and easy to use. RESULTS: The performance and reliability of the method are demonstrated through three case studies: Firstly, on synthetic images to study the behavior and assess the relevance of the algorithm in diverse situations and hence, in various potential fields. Secondly, on histological images derived from experimental chitosan-platelet-rich-plasma hybrid biomaterial, where the quantitative results are compared to a qualitative classification provided by an expert in the field. Thirdly, on experimental nanocomposites images for which results are compared to two other homogeneity quantification algorithms from the field of nanocomposites. CONCLUSION AND SIGNIFICANCE: By quantifying homogeneity, the MASQH method may help to compare disparate studies in the literature and quantitatively demonstrate the impact of homogeneity in various fields. The MASQH method is freely available online.

Poly(2-Propylacrylic Acid) Increases In Vitro Bioactivity of Chitosan/mRNA Nanoparticles.

Chitosan-based nanoparticles have been extensively studied for the delivery of nucleic acids. Previous results suggest that these nanoparticles have limited ability to escape the endosome, one of the main cellular barriers hindering nucleic acid delivery. Escape can be improved by the addition of endosomolytic agents during the formulation process or by developing delivery systems with intrinsic properties to disrupt endosomal membranes. In this study, Poly(2-Propylacrylic Acid) (PPAA), an anionic synthetic polymer with known membrane lytic activity was added to the binary chitosan/mRNA nanoparticles to improve bioactivity. The ionization behavior of PPAA was characterized to identify conditions in which PPAA is sufficiently charged to interact electrostatically with chitosan and thus form nanoparticles. The physicochemical characteristics (hydrodynamic diameter, polydispersity index, ζ-potential) and the in vitro transfection efficiency (bioactivity) of this new family of CS/mRNA/PPAA ternary nanoparticles were evaluated. The addition of PPAA to CS/mRNA nanoparticles was shown to be an efficient strategy to augment in vitro bioactivity. The optimal formulation reached an expression level  ~86% of the commercial lipid control at pH 6.5 without any signs of metabolic toxicity. In this paper, we report the effect of salt and pH on the ionization behavior of PPAA and demonstrate 1) successful incorporation of PPAA into/onto nanoparticles, 2) improved bioactivity with PPAA, and 3) that the kosmotropic effects of trehalose play a minimal role in the apparent increase in bioactivity in presence of trehalose.

Efficiency of Chitosan/Hyaluronan-Based mRNA Delivery Systems In Vitro: Influence of Composition and Structure.

Messenger RNA (mRNA)-containing nanoparticles were produced by electrostatic complexation with a library of pharmaceutical grade chitosans with different degrees of deacetylation and hyaluronic acids (HAs) (native vs. sulfated). Polymer length (Mn), HA degree of sulfation (DS), and amine to phosphate to carboxyl + sulfate (from HA) ratio (N:P:C) were controlled. In vitro transfections were performed in the presence/absence of trehalose and at different pH. Particle size and ζ-potential were correlated with transfection efficiency. Polymer length and charge densities (degree of deacetylation, degree of sulfation) of both HA and chitosan had a direct influence on transfection efficiency through modulation of avidity to mRNA. N:P:C ratio, trehalose, mixing concentration, and nucleic acid dose influenced transfection efficiency with optimized formulations reaching ∼60%-65% transfection efficiency relative to commercially available lipid control with no apparent toxicity for transfection at slightly acidic pH 6.5.

A novel image analysis algorithm reveals that media conditioned with chitosan and platelet-rich plasma biomaterial dose dependently increases fibroblast migration in a scratch assay.

Chitosan (CS) and Platelet-Rich Plasma (PRP) both display interesting properties for wound healing applications. A hybrid CS-PRP biomaterial was previously developped, consisting of a freeze dried CS formulation solubilized in PRP that promotes tissue repair and regeneration. The purpose of the current study was to investigate the ability of the CS-PRP biomaterial to stimulate cell migrationin vitro. Scratch assays revealed that CS-PRP significantly stimulates the migration rate of cells compared to cells in culture medium but not differently than PRP alone. The increase in the migration rate is dose-dependent at low dose and reaches a plateau corresponding with maximum cell motility. Cell migration rate as a function of the number of platelets that have degranulated in culture medium (to which total concentration of growth factors contributing to cell response is proportionnal), follows a modified Hill model. To analyze photographs taken during the assay and follow cell migration, an open source image analysis algorithm was developed: SAMScratch (Systematic Area Measurement of Scratch - available here:https://github.com/Biomaterials-and-Cartilage-Laboratory/SAM-Scratch). Compared with other existing analysis tools, the algorithm is precise in the determination of the scratch area and performs equally well with usual and challenging images. This study resulted in the creation of a freely available application for scratch assay analysis and provided evidence that CS-PRP implants hold promise for treatment of wounds.

Multiple platelet-rich plasma preparations can solubilize freeze-dried chitosan formulations to form injectable implants for orthopedic indications.

BACKGROUND: Platelet-rich plasma (PRP) has been used to solubilize freeze-dried chitosan (CS) formulations to form injectable implants for tissue repair. OBJECTIVE: To determine whether the in vitro performance of the formulations depends on the type of PRP preparation used to solubilize CS. METHODS: Formulations containing 1% (w/v) CS with varying degrees of deacetylation (DDA 80.5-84.8%) and number average molar mass (Mn 32-55 kDa), 1% (w/v) trehalose and 42.2 mM calcium chloride were freeze-dried. Seven different PRP preparations were used to solubilize the formulations. Controls were recalcified PRP. RESULTS: CS solubilization was achieved with all PRP preparations. CS-PRP formulations were less runny than their corresponding PRP controls. All CS-PRP formulations had a clotting time below 9 minutes, assessed by thromboelastography, while the leukocyte-rich PRP controls took longer to coagulate (>32 min), and the leukocyte-reduced PRP controls did not coagulate in this dynamic assay. In glass culture tubes, all PRP controls clotted, expressed serum and retracted (43-82% clot mass lost) significantly more than CS-PRP clots (no mass lost). CS dispersion was homogenous within CS-PRP clots. CONCLUSIONS: In vitro performance of the CS-PRP formulations was comparable for all types of PRPs assessed.

siRNA Delivery with Chitosan: Influence of Chitosan Molecular Weight, Degree of Deacetylation, and Amine to Phosphate Ratio on in Vitro Silencing Efficiency, Hemocompatibility, Biodistribution, and in Vivo Efficacy.

Chitosan (CS) shows in vitro and in vivo efficacy for siRNA delivery but with contradictory findings for incompletely characterized systems. For understanding which parameters produce effective delivery, a library of precisely characterized chitosans was produced at different degrees of deacetylation (DDAs) and average molecular weights (Mn). Encapsulation and transfection efficiencies were characterized in vitro. Formulations were selected to examine the influence of Mn and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection. Hemocompatibility and in vivo biodistribution were then investigated for different Mn, N:P ratios, and doses. Nanoparticle uptake and gene silencing correlated with increased surface charge, which was obtained at high DDA and high Mn. A minimum polymer length of ∼60-70 monomers (∼10 kDa) was required for stability and knockdown. In vitro knockdown was equivalent to lipid control with no metabolic or genotoxicity. An inhibitory effect of serum on biological performance was dependent on DDA, Mn, and N:P. In vivo biodistribution in mice show accumulation of nanoparticles in kidney with 40-50% functional knockdown.

Lyophilisation and concentration of chitosan/siRNA polyplexes: Influence of buffer composition, oligonucleotide sequence, and hyaluronic acid coating.

Chitosan (CS)/siRNA polyplexes have great therapeutic potential for treating multiple diseases by gene silencing. However, clinical application of this technology requires the development of concentrated, hemocompatible, pH neutral formulations for safe and efficient administration. In this study we evaluate physicochemical properties of chitosan polyplexes in various buffers at increasing ionic strengths, to identify conditions for freeze-drying and rehydration at higher doses of uncoated or hyaluronic acid (HA)-coated polyplexes while maintaining physiological compatibility. Optimized formulations are used to evaluate the impact of the siRNA/oligonucleotide sequence on polyplex physicochemical properties, and to measure their in vitro silencing efficiency, cytotoxicity, and hemocompatibility. Specific oligonucleotide sequences influence polyplex physical properties at low N:P ratios, as well as their stability during freeze-drying. Nanoparticles display greater stability for oligodeoxynucleotides ODN vs siRNA; AT-rich vs GC-rich; and overhangs vs blunt ends. Using this knowledge, various CS/siRNA polyplexes are prepared with and without HA coating, freeze-dried and rehydrated at increased concentrations using reduced rehydration volumes. These polyplexes are non-cytotoxic and preserve silencing activity even after rehydration to 20-fold their initial concentration, while HA-coated polyplexes at pH∼7 also displayed increased hemocompatibility. These concentrated formulations represent a critical step towards clinical development of chitosan-based oligonucleotide intravenous delivery systems.

Stability and binding affinity of DNA/chitosan complexes by polyanion competition.

The stability of DNA/chitosan complexes upon exposure to hyaluronic acid, chondroitin sulfate, and heparin, was assessed by fluorescence spectroscopy to quantify DNA release. Only the highly charged heparin was found to release DNA from the complexes. Complex stability upon exposure to heparin increased with the degree of deacetylation and molecular weight of chitosan and with the ratio of chitosan amino groups to DNA phosphate groups (N/P ratio) in the complexes. Isothermal titration microcalorimetry revealed that among polyanions tested, only heparin has a binding affinity to chitosan approaching that of DNA and can therefore release DNA from the complexes. These results also indicate that anionic components with sufficiently high charge density can induce extracellular or intracellular release of DNA, the former negatively affecting delivery efficiency while the latter is required for gene transfer to occur. Our findings also suggest that increased N/P ratio of the complexes can play an important role in preventing premature dissociation of DNA/polycation complexes upon interaction with anionic components in extracellular milieu.

Automated in-line mixing system for large scale production of chitosan-based polyplexes.

Chitosan (CS)-based polyplexes are efficient non-viral gene delivery systems that are most commonly prepared by manual mixing. However, manual mixing is not only poorly controlled but also restricted to relatively small preparation volumes, limiting clinical applications. In order to overcome these drawbacks and to produce clinical quantities of CS-based polyplexes, a fully automated in-line mixing platform was developed for production of large batches of small-size and homogeneous CS-based polyplexes. Operational conditions to produce small-sized homogeneous polyplexes were identified. Increasing mixing concentrations of CS and nucleic acid was directly associated with an increase in size and polydispersity of both CS/pDNA and CS/siRNA polyplexes. We also found that although the speed of mixing has a negligible impact on the properties of CS/pDNA polyplexes, the size and polydispersity of CS/siRNA polyplexes are strongly influenced by the mixing speed: the higher the speed, the smaller the size and polydispersity. While in-line and manual CS/pDNA polyplexes had similar size and PDI, CS/siRNA polyplexes were smaller and more homogenous when prepared in-line in the non-laminar flow regime compared to manual method. Finally, we found that in-line mixed CS/siRNA polyplexes have equivalent or higher silencing efficiency of ApoB in HepG2 cells, compared to manually prepared polyplexes.

Lysosomal rupture induced by structurally distinct chitosans either promotes a type 1 IFN response or activates the inflammasome in macrophages.

Chitosan is a family of glucosamine and N-acetyl glucosamine polysaccharides with poorly understood immune modulating properties. Here, functional U937 macrophage responses were analyzed in response to a novel library of twenty chitosans with controlled degree of deacetylation (DDA, 60-98%), molecular weight (1 to >100 kDa), and acetylation pattern (block vs. random). Specific chitosan preparations (10 or 190 kDa 80% block DDA and 3, 5, or 10 kDa 98% DDA) either induced macrophages to release CXCL10 and IL-1ra at 5-50 µg/mL, or activated the inflammasome to release IL-1ß and PGE2 at 50-150 µg/mL. Chitosan induction of these factors required lysosomal acidification. CXCL10 production was preceded by lysosomal rupture as shown by time-dependent co-localization of galectin-3 and chitosan and slowed autophagy flux, and specifically depended on IFN-ß paracrine activity and STAT-2 activation that could be suppressed by PGE2. Chitosan induced a type I IFN paracrine response or inflammasome response depending on the extent of lysosomal rupture and cytosolic foreign body invasion. This study identifies the structural motifs that lead to chitosan-driven cytokine responses in macrophages and indicates that lysosomal rupture is a key mechanism that determines the endogenous release of either IL-1ra or IL-1ß.

Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery.

Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.

Preparation of Concentrated Chitosan/DNA Nanoparticle Formulations by Lyophilization for Gene Delivery at Clinically Relevant Dosages.

Chitosan/DNA polyplexes have been optimized for efficient and safe in vitro and in vivo gene delivery. Clinical application of this technology requires the development of formulations with higher concentrations to reach therapeutic dosages. Polyplexes were prepared using chitosan and EGFPLuc plasmids. Freeze-thawing and freeze-drying studies were performed to identify and optimize lyoprotectant and buffer contents in formulations. Freeze-dried samples were rehydrated in reduced volumes to increase their final DNA dose. Nanoparticle physicochemical properties were analyzed, and their transfection efficiency and cytotoxicity were measured in human embryonic kidney 293 cells. Data showed that 3.5 mM histidine buffer (pH 6.5) combined with one of 0.5% wt/vol sucrose, dextran 5 kDa, or trehalose was required to prevent polyplex aggregation during freeze-drying. Optimal formulations could be concentrated 20-fold, to a clinically desired ∼1 mg of DNA/mL, while maintaining near physiological pH and tonicity. Polyplexes were predominantly spherical, with diameters below 200 nm, polydispersity indexes below 0.32, and zeta potentials above +19 mV. Rehydrated formulations had transfection efficiencies no less than 65% of fresh polyplexes without excipients and had no effect on viability and metabolic activity of human embryonic kidney 293 cells. These concentrated formulations represent an important step toward clinical use of chitosan-based gene delivery systems.

Combined analysis of polycation/ODN polyplexes by analytical ultracentrifugation and dynamic light scattering reveals their size, refractive index increment, stoichiometry, porosity, and molecular weight.

Analytical ultracentrifugation (AUC) and dynamic light scattering (DLS) were combined to characterize polyplexes formed with 10 kDa chitosan or 10 kDa PEI and oligodeoxynucleotides (ODN). Combined analysis revealed that both polyplexes were highly porous (over 80%) and that their weight average hydrodynamic diameters were of 46 and 55 nm for chitosan/ODN and PEI/ODN complexes, respectively. Transformation of the sedimentation coefficient distribution to a size and molecular weight distribution gave an average molecular weight of 19 and 29 MDa for chitosan and PEI polyplexes, respectively. Data from AUC also allowed for the calculation of the actual dn/dc and N/P ratios of each polyplex. Additional data from scanning electron microscopy and static light scattering confirmed the conclusions that were initially derived from AUC and DLS, thus validating that the combination of AUC and DLS is a powerful approach to characterize polyplexes in terms of refractive index increment, size, and molecular weight distributions, as well as porosity.