Exosomes isolated from two different cell lines using three different isolation techniques show variation in physical and molecular characteristics.

Exosomes are the nanoscopic lipid bi-layered extracellular vesicles with the potential to be utilized as targeted therapeutics. In our investigation, we compared three major exosome isolation techniques that were Total Exosome Isolation reagent (TEI), Protein organic solvent precipitation (PROSPR) and differential ultracentrifugation (UC) based on the biophysical and physicochemical characteristics of exosomes isolated from COLO 205 and MCF-7 cancer cell's conditioned media with an aim to select a suitable method for translational studies. 3D image analysis and particle size distribution of exosomes from their HRTEM images depicted the morphological differences. Molecular and analytical characterization of exosomes using western blotting, Raman and ATR-FTIR spectroscopy and the multivariate analysis on the spectral data obtained, assessed for better molecular specifications and purity of particle. TEI method isolated exosomes with higher exosomal yield, purity, and recovery directly translatable into drug delivery and targeted therapeutics whereas ultracentrifuge had good recovery of particle morphology but showed particle aggregation and yielded exosomes with smaller mean size. PROSPR technique isolated a mixture of EVs, showed lower protein recovery in PAGE and western blotting but higher spectroscopic protein to lipid ratio and distinguishable EV population in multivariate analysis compared to exosomes isolated by TEI and UC. This comparative study should help in choosing a specific exosome isolation technique required for the objective of downstream applications.

Lipids from algal biomass provide new (nonlamellar) nanovectors with high carrier potentiality for natural antioxidants.

Lipid mesophases are lyotropic liquid crystalline systems which differ from liposomes and other globular aggregates in dilute regimes due to their inner ordering. It is known that natural lipids enable to obtain a rich variety of nanosystems and many of them have been proposed as delivery agents for bioactive compounds. Due to their packing parameters, several classes of lipids found in natural sources are able to self-assemble into nonlamellar structures. Among lipids occurring in plants and algae, triglycerides display this tendency. In the present study we examine new nanosystems built with lipids extracted from the marine microalga Nannochloropsis sp and their use as carriers for lipophilic antioxidants. The antioxidants studied, curcumin and tocopherol were encapsulated with high rate in the carriers. The physico-chemical characterization of plain and loaded vectors showed their structure and localization site, as well as the structure-functionality relationship related to potential drug delivery. The results show that the cargo molecules play an active role in driving the interactions which characterize the overall structure of the aggregates. The systems studied showed several coexisting mesophases, the most predominant structure being of cubic symmetry.

Transdermal delivery enhancement of carvacrol from Origanum vulgare L. essential oil by microemulsion.

Carvacrol has been reported for analgesic and anti-inflammatory activity by cyclooxygenase inhibition but it could induce gastrointestinal toxicity because of its non-selective inhibition. Therefore, the present study aimed to develop transdermal microemulsion from Origanum vulgare essential oil to deliver carvacrol into and through the skin which would overwhelm the gastrointestinal problems. O. vulgare essential oil was extracted by hydrodistillation and its carvacrol content was determined using high performance liquid chromatography. Pseudoternary phase diagrams were constructed using water dilution method to investigate the suitable microemulsion components. Microemulsions were then characterized for external appearance, particle size, size distribution, zeta potential, electrical conductivity, refractive index, viscosity, transmittance, pH, and stability. Additionally, the irritation property of microemulsions were investigated by hen's egg on the chorioallantoic membrane assay. The release profile, percutaneous absorption, and skin retention were investigated using dialysis bag and Franz diffusion cell, respectively. The interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were investigated using the enzyme-linked immunosorbent assay. The results remarked that carvacrol was a major component of O. vulgare essential oil with high concentration of 83.7%. The most suitable microemulsion (ME 1), composing of 5% w/w O. vulgare essential oil, 25%w/w Tween 60, 25%w/w butylene glycol, and 45%w/w deionized water, had the smallest internal droplet size (179.5 ± 27.9 nm), the narrowest polydispersity index (0.30 ± 0.07), the highest transmittance (93.13 ± 0.04%), and Newtonian flow behavior with low viscosity of 0.30 ± 0.07 Pas. ME 1 could reduce the irritation effect of O. vulgare essential oil since ME 1 (IS = 3.1 ± 0.10) exhibited significantly lower irritation effect than its blank formulation (IS = 4.8 ± 0.02) and O. vulgare oil solution (IS = 5.0 ± 0.01) (p < 0.05). Furthermore, ME 1 sustain released carvacrol from the formulation, remarkedly deliver more carvacrol through the skin layer (2.6 ± 2.2%) and significantly retained carvacrol in the skin layer (2.60 ± 1.25%). Additionally, ME 1 significantly enhanced IL-6 inhibition of O. vulgaris oil and carvacrol (p < 0.05). Therefore, O. vulgaris oil microemulsion was suggested to be used for the transdermal delivery and anti-inflammatory activities enhancement of carvacrol.

Naturally Drug-Loaded Chitin: Isolation and Applications.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Screening and characterization of a novel high-efficiency tumor-homing cell-penetrating peptide from the buffalo cathelicidin family.

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell-penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo-derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell-penetrating antimicrobial peptides, including the well-known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC-7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC-5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia-derived type-I ribosome-inactivating protein MAP 30, and the cytotoxicity of the MAP 30-CAT fusion protein in the tumor cell line SMMC-7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30-CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC-5 was approximately twofold higher than the value for SMMC-7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38-fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor-homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.

Influence of Manothermosonication on the Physicochemical and Functional Properties of Ferritin as a Nanocarrier of Iron or Bioactive Compounds.

Ferritin is a multisubunit protein with a hollow interior interface and modifiable surfaces. In this study, the manothermosonication (MTS) technology was applied to apo-red bean seed ferritin (apoRBF) to produce the MTS-treated apoRBF (MTFS). MTS treatment (200 kPa, 50 °C, and 40 s) maintained the spherical morphology of apoRBF (12 nm), but reduced the content of α-helix structure and increased the content of random coil structure, and correspondingly decreased the ferritin stability. The MTS treatment also affected the ferritin's iron storage function by decreasing its iron oxidative deposition activity and increasing the iron release activity. Importantly, the disassembly and reassembly properties of the MTFS induced by pH changes were retained, which facilitated its usage in encapsulation of tea polyphenol-epigallocatechin gallate (EGCG) into the ferritin by a relatively benign pH conversion routine (pH 3.0/6.8). In addition, the water solubility of the MTFS was increased, leading to the improved encapsulation efficiency of the EGCG molecules. This study will facilitate the ferritin modification and functionalization by MTS to design a protein variant to be used as new scaffold for iron and bioactive compounds.

Design strategies and application progress of therapeutic exosomes.

Exosomes have great potential to be drug delivery vehicles due to their natural material transportation properties, intrinsic long-term circulatory capability, and excellent biocompatibility, which are suitable for delivering a variety of chemicals, proteins, nucleic acids, and gene therapeutic agents. However, an effective method of loading specific protein agents into exosomes for absorption by target cells is still lacking. The application potential of exosome is still limited. In this review, we discussed the methods for loading specific treating molecules (proteins, nucleic acids and small chemicals) into exosomes, the design strategies for cell and tissue targeting, and the factors for exosome formation. This review can be used as a reference for further research as well as for the development of therapeutic exosomes.

Inulin: A novel and stretchy polysaccharide tool for biomedical and nutritional applications.

Inulin (INU) is a flexible, fructan type polysaccharide carbohydrate, mainly obtained from the root of chicory. It is a water-soluble dietary fibre and has been recently approved by the Food and Drug Administration for improving the nutritional values of food products. INU is not digested or fermented in the initial portion of the human digestive system and directly reaches on the distal portion of the colon. Owing to this superior property, INU is specially applied to develop specific carrier systems for localized delivery of drugs related to colon diseases. Several studies proved that the fermented bi-products of INU help the growth and stimulating activity of colon bacteria e.g. Bifidobacterium and Lactobacilli. INU also has several inherent therapeutic effects like reduction of tumor risks, help in calcium ion absorption, anti-inflammatory, antioxidant properties etc. Apart from these, INU has been used for different pharmaceutical applications as a drug carrier, stabilizing agent, cryoprotectant, and an alternative to fats and sugars. Here, we review the applications of INU in different areas of biomedical science, look back into the nutritional effects of INU and outline various routes of administration of INU-based formulations.

Microcarriers Based on Glycosaminoglycan-Like Marine Exopolysaccharide for TGF-ß1 Long-Term Protection.

Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation or due to aging are thus irreversible and may induce the loss of the joint function. To repair cartilaginous defects, tissue engineering approaches are under intense development. Association of cells and signalling proteins, such as growth factors, with biocompatible hydrogel matrix may lead to the regeneration of the healthy tissue. One current strategy to enhance both growth factor bioactivity and bioavailability is based on the delivery of these signalling proteins in microcarriers. In this context, the aim of the present study was to develop microcarriers by encapsulating Transforming Growth Factor-ß1 (TGF-ß1) into microparticles based on marine exopolysaccharide (EPS), namely GY785 EPS, for further applications in cartilage engineering. Using a capillary microfluidic approach, two microcarriers were prepared. The growth factor was either encapsulated directly within the microparticles based on slightly sulphated derivative or complexed firstly with the highly sulphated derivative before being incorporated within the microparticles. TGF-ß1 release, studied under in vitro model conditions, revealed that the majority of the growth factor was retained inside the microparticles. Bioactivity of released TGF-ß1 was particularly enhanced in the presence of highly sulphated derivative. It comes out from this study that GY785 EPS based microcarriers may constitute TGF-ß1 reservoirs spatially retaining the growth factor for a variety of tissue engineering applications and in particular cartilage regeneration, where the growth factor needs to remain in the target location long enough to induce robust regenerative responses.

Preparation and anti-tumor efficiency evaluation of bacterial magnetosome-anti-4-1BB antibody complex: Bacterial magnetosome as antibody carriers isolated from Magnetospirillum gryphiswaldense.

Bacterial magnetosomes (BMs) are used as carriers for antibodies, enzymes, and nucleic acids. This study aimed to demonstrate the clinical utility of BMs derived from Magnetospirillum gryphiswaldense for use in anti-tumor immunotherapy. Bis(sulfosuccinimidyl) suberate (BS3) was used to prepare BM-anti-4-1BB antibody complexes. We used syngeneic TC-1 mouse models of cancer to investigate whether BMs combined with an anti-4-1BB agonistic antibodies could enhance the therapeutic effects of anti-4-1BB antibodies in localized disease settings. Anti-4-1BB antibodies were combined with purified BMs at a concentration of 168 mg antibody per milligram BM (mg IgG/mg BM) using BS3. The anti-4-1BB antibody-coupled BMs (BM-Ab complexes) and control BMs displayed similar morphologies and measurements when examined by transmission electron microscope (TEM). In a mouse tumor model, intravenous injection of BM-Abs combined with magnetic treatment resulted in greater tumor protection than did other treatment methods (P < 0.05). These results demonstrate the in vivo anti-tumor properties of BM-Abs complexes. The coupling of anti-4-1BB antibodies to magnetosomes may have potential for clinical application to antitumor antibody therapy.

Evaluation of novel microcrystalline cellulose from Ensete glaucum (Roxb.) Cheesman biomass as sustainable drug delivery biomaterial.

Microcrystalline cellulose (MCC) is one of the most important functional excipient in Pharmaceutical industries. A renewable biomass from Ensete glaucum (Roxb.) was investigated as a potential source of a novel functional MCC. MCC was prepared by a simple, dilute acid hydrolysis and characterized through FTIR, DSC, XRD, along with micromeritic studies. Functional properties such as packing, rearrangement, consolidation and compactibility of the prepared MCC were also evaluated in view of its application as drug delivery biomaterial. Results suggest that the prepared MCC exhibit properties comparable to commercially available standard MCC. From Kawakita and Heckel plots, it was observed that the new MCC consolidates better than the standard MCC. Disintegration efficiency test also indicates that the novel MCC functions as a better tablet disintegrant to the standard MCC indicating the potential of Ensete glaucum (Roxb.) as a green resource for preparation of the low cost, functional and sustainable carbohydrate polymer.

Extraction, characterization, utilization as wound dressing and drug delivery of Bletilla striata polysaccharide: A review.

This review provides updated information on a kind of active polysaccharides extracting from Chinese traditional herb Bletilla striata. Preliminary investigations have listed several isolation approaches of extracting Bletilla striata polysaccharide (BSP) and the characterization result showed that the backbone of BSP has mainly consisted of 1,4-linked mannosyl residues and 1,4-linked glucosyl residues. Remarkably, this review sums up the exploitation of BSP as biomaterials, including the preparation, bioactivity and effect mechanism of BSP-based wound dressings and drug deliveries. BSP exhibits excellent healing function mainly due to its modulation of macrophages throughout inflammation and proliferation periods. BSP-based drug vehicles include micelles, nanoparticles, microspheres and microneedles which display anti-cancer functions of targeted delivering drugs and drug capability of itself as well. This review aims to pave the way for further exploitation of this compound in biomedical area.

Simultaneous two drugs release form Janus particles prepared via polymerization-induced phase separation approach.

Seeded emulsion polymerization of 2-dimethylaminoethylamino methacrylate (DMAEMA) was carried out using monodispersed poly(2-hydroxyehtyl methacrylate) (PHEMA) seeds to produce Janus particles. Three feeding approaches were used comprising one together, rest and continuous feeding methods to investigate different morphologies. However, FE-SEM results showed that all feeding approaches yielded dumbbell-like Janus particles. Furthermore, snowman-like Janus particles were obtained via seeded distillation precipitation polymerization (DPP). It is shown that minimizing the total interfacial free energy alongside difference in solubility parameters of Janus domains are responsible for obtained morphologies. Two different morphologies (dumbbell-like and snowman-like) were chosen as carriers of ibuprofen and DOX simultaneously. Also, simultaneous release of two drugs were investigated in different conditions. Dumbbell-like Janus particles showed higher ibuprofen loading whereas DOX was more loaded onto snowman-like Janus particles. Also, DOX was released more rapidly through Janus particles at different pH values and both types of Janus particles showed similar drugs release behaviors.

Isolation and characterization of natural polysaccharide from Cassia Obtustifolia seed mucilage as film forming material for drug delivery.

In this work Cassia obtustifolia seed mucilage isolated and evaluated as novel excipient for drug delivery. Seed mucilage was evaluated qualitatively and quantitatively for presence of polysaccharide. A novel biodegradable film based on mucilage obtained from seeds of Cassia obtustifolia was fabricated and characterized. The microstructure, mechanical and thermal properties of the film were determined. Results of the scanning electron microscopy revealed a smooth and regular surface morphology. DSC and X-ray diffraction studies revealed an amorphous structure of Cassia obtustifolia seed mucilage films. In vitro degradation simulated body fluids and oral acute toxicity studies with high LD50 value of >2 g/kg of body weight demonstrate its safety as excipient. Diclofenac loaded film exhibited sustained drug release due to swelling and diffusion of film. These findings demonstrated that the Cassia obtustifolia seed mucilage had potential to use as film forming excipient with enhanced characteristics for drug delivery application.

Nucleic acid loading and fluorescent labeling of isolated extracellular vesicles requires adequate purification.

Extracellular vesicles (EVs) are nanosized vesicular structures released by cells to communicate with one another. The growing interest in the (patho)physiological function and potential pharmaceutical application of these vesicles is accompanied by a vast number of new research groups entering this research field and a plethora of different protocols to separate EVs from non-vesicular components. This lack of uniformity often generates conflicting or difficult-to-compare results. Here we provide a comparative analysis of different EV isolation strategies, discussing the purity of the final isolate and highlighting the importance of purity on downstream experimental readouts. First, we show that ultracentrifugation (UC) of B16F10 melanoma cell-derived conditioned medium co-purifies proteins or protein complexes with nuclease activity. Such contaminants should be taken into account when aiming to apply EVs as delivery carriers for exogenous nucleic acids. Second, three commonly used purification strategies (i.e. precipitation, UC and density-gradient centrifugation) were evaluated for their ability to remove non-incorporated fluorescent dye (i.e. the lipophilic PKH67 dye), important when probing EV interactions with cells. For both types of impurities, endogenous and exogenous, density gradient purification outperforms the other evaluated methods. Overall, these results demonstrate that the implementation of stringent purification protocols and adequate controls is of pivotal importance to draw reliable conclusions from downstream experiments performed with EV isolates.

Xyloglucan: A functional biomacromolecule for drug delivery applications.

The near future of drug delivery system would lie in the search of a versatile and innocuous material, based mostly on the natural resources. The tamarind seed xyloglucan (XG) is a natural neutral hemicellulose and a hydrophilic polysaccharide consisting of a main chain of glucan backbone with xylose and galactose side chains. XG is endowed with idiosyncratic mucoadhesive and in situ gelling properties which rated XG as an attractive, functional polymer for numerous drug delivery applications. In milieu of this, the present review is designed to underline the plausible potential of XG or XG-based systems in drug delivery. The feasibility of surface-tailoring, the flexibility of chemical-modification, and the possibility as ligand-conjugations grant XG an extraordinary consideration in the scientific territory. The authors are hopeful that the versatility of XG would meet the expectations of regulatory authorities and the XG-based products will serve the therapeutic needs of the community in the future, if sufficiently investigated and promising outcomes are obtained in human subjects.

Zein-based Nanocarriers as Potential Natural Alternatives for Drug and Gene Delivery: Focus on Cancer Therapy.

Protein nanocarriers possess unique merits including minimal cytotoxicity, numerous renewable sources, and high drug-binding capability. In opposition to delivery carriers utilizing hydrophilic animal proteins, hydrophobic plant proteins (e.g, zein) have great tendency in fabricating controlled-release particulate carriers without additional chemical modification to stiffen them, which in turn evades the use of toxic chemical crosslinkers. Moreover, zein is related to a class of alcohol-soluble prolamins and generally recognized as safe (GRAS) carrier for drug delivery. Various techniques have been adopted to fabricate zein-based nanoparticulate systems including phase separation coacervation, spray-drying, supercritical anti-solvent approach, electrospinning and self-assembly. This manuscript reviews the recent advances in the zein-based colloidal nano-carrier systems such as nanospheres, nanocapsules, micelles and nanofibers with a special focus on their physicochemical characteristics and drug delivery applications.

Sulfated archaeal glycolipid archaeosomes as a safe and effective vaccine adjuvant for induction of cell-mediated immunity.

Archaeosomes are liposomal vesicles composed of ether glycerolipids unique to the domain of Archaea. Unlike conventional ester-linked liposomes, archaeosomes exhibit high stability and possess strong adjuvant and immunostimulatory properties making them an attractive vaccine delivery vehicle. Traditionally comprised of total polar lipids (TPL) or semi-synthetic phospho-glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups, archaeosomes can induce robust and long-lasting humoral and cell-mediated immune responses against antigenic cargo and provide protection in murine models of infectious disease and cancer. However, traditional TPL archaeosome formulations are relatively complex comprising several lipid species. Semi-synthetic archaeosomes tested previously contain a combination of several phospho-glycolipids (negative and neutral charged) to produce a stable, uniform-sized liposome formulation. Moreover, they involve many synthetic steps to arrive at the final desired glycolipid composition. Herein, we present a novel adjuvant formulation comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA). SLA individually or mixed with uncharged glyolipid (lactosylarchaeol, LA) constituted efficacious carrier vesicles for entrapped antigens (ovalbumin or melanoma associated tyrosinase-related protein 2 [TRP-2]) and induction of strong cell-mediated responses in mice and protection against subsequent B16 melanoma tumor challenge. Thus, semi-synthetic sulfated glycolipid archaeosomes represent a new class of adjuvants that will potentially ease manufacturing and scale-up, while retaining immunostimulatory activity.

Yield Optimisation of Hepatitis B Virus Core Particles in E. coli Expression System for Drug Delivery Applications.

An E. coli expression system offers a mean for rapid, high yield and economical production of Hepatitis B Virus core (HBc) particles. However, high-level production of HBc particles in bacteria is demanding and optimisation of HBc particle yield from E. coli is required to improve laboratory-scale productivity for further drug delivery applications. Production steps involve bacterial culture, protein isolation, denaturation, purification and finally protein assembly. In this study, we describe a modified E. coli based method for purifying HBc particles and compare the results with those obtained using a conventional purification method. HBc particle morphology was confirmed by Atomic Force Microscopy (AFM). Protein specificity and secondary structure were confirmed by Western Blot and Circular Dichroism (CD), respectively. The modified method produced ~3-fold higher yield and greater purity of wild type HBc particles than the conventional method. Our results demonstrated that the modified method produce a better yield and purity of HBc particles in an E. coli-expression system, which are fully characterised and suitable to be used for drug delivery applications.

Analysis of chitosan/tripolyphosphate micro- and nanogel yields is key to understanding their protein uptake performance.

Chitosan/tripolyphosphate (TPP) micro- and nanogels are widely explored as vehicles for protein drug and vaccine delivery. Yet, aside from the consensus that protein uptake into these particles is enhanced by stronger protein/particle binding, factors that control their uptake performance, such as differences in the chitosan, TPP and protein concentrations, remain poorly understood. Here, we show that many of the differences in the reported association efficiencies (AE-values) for protein uptake likely reflect the largely-ignored variability in the particle yield (XAgg), which is the fraction of the added chitosan that self-assembles into particles and (like the AE) varies with the chitosan, TPP and protein concentrations. Factors affecting XAgg are first systematically explored. The AE is then shown to scale almost linearly with the XAgg (which increases with the TPP and protein-to-chitosan ratios) until all chitosan aggregates into particles. Remarkably, the data collected at variable TPP and protein concentrations collapses onto a single AE∝XAgg curve for each protein type. Further analysis of protein/particle binding reveals this rise in AE with XAgg to reflect: (1) an increase in binding sites within the particles; and (2) a decrease in soluble (non-particulate) chitosan molecules, which form soluble protein/chitosan complexes and compete with the chitosan/TPP particles for the unassociated protein. These findings highlight the need to carefully analyze the effects of formulation parameters on chitosan/TPP particle yields and can likely be extended to other ionically crosslinked colloidal drug carriers.