ROS-responsive glycol chitosan-linked prodrug nanoparticle as a nanoplatform for tumor chemo-photodynamic therapy.

Herein, we designed and synthesized novel reactive oxygen species (ROS)-responsive glycol chitosan-doxorubicin (DOX) prodrug via a ROS-cleavable thioketal (TK) linker. The obtained GC-TK-DOX formed self-assembled nanoparticles of 312 nm in aqueous media. Photosensitizers zinc phthalocyanine (ZnPc)-loaded GC-TK-DOX (GC-TK-DOX/ZnPc) nanoparticles were fabricated by using a dialysis approach. The GC-TK-DOX and GC-TK-DOX/ZnPc nanoparticles were nearly spherical by transmission electron microscopy (TEM) observation. Under 660-nm laser irradiation, GC-TK-DOX/ZnPc could generate singlet oxygen. Further, GC-TK-DOX/ZnPc nanoparticles exhibited ROS-sensitive release of DOX and ZnPc in vitro. GC-TK-DOX/ZnPc with laser irradiation showed more drug uptake and higher cytotoxic effects than GC-TK-DOX/ZnPc without irradiation, free DOX and GC-TK-DOX in HeLa tumor cells. Overall, these findings suggested that GC-TK-DOX/ZnPc could be a promising nanoarchitecture for synergetic chemo-photodynamic therapy against tumors.

Aurozyme: A Revolutionary Nanozyme in Colitis, Switching Peroxidase-Like to Catalase-Like Activity.

A therapeutic strategy that could address colitis of multiple etiologies while restoring the dysbiosis of gut microbiota is attractive. Here, Aurozyme, a novel nanomedicine comprised of gold nanoparticles (AuNPs) and glycyrrhizin (GL) with a glycol chitosan coating layer, as a promising approach for colitis, is demonstrated. The unique feature of Aurozyme is the conversion of harmful peroxidase-like activity of AuNPs to beneficial catalase-like activity due to the amine-rich environment provided by the glycol chitosan. This conversion process enables Aurozyme to oxidize the hydroxyl radicals derived from AuNP, producing water and oxygen molecules. In fact, Aurozyme effectively scavenges reactive oxygen/reactive nitrogen species (ROS/RNS) and damage-associated molecular patterns (DAMPs), which can attenuate the M1 polarization of macrophage. It exhibits prolonged adhesion to the lesion site, promoting sustained anti-inflammatory effects and restoring intestinal function in colitis-challenged mice. Additionally, it increases the abundance and diversity of beneficial probiotics, which are essential for maintaining microbial homeostasis in the gut. The work highlights the transformative potential of nanozymes for the comprehensive treatment of inflammatory disease and represents an innovative switching technology of enzyme-like activity by Aurozyme.

In vivo toxicity evaluation of tumor targeted glycol chitosan nanoparticles in healthy mice: repeated high-dose of glycol chitosan nanoparticles potentially induce cardiotoxicity.

BACKGROUND: Glycol chitosan nanoparticles (CNPs) have emerged as an effective drug delivery system for cancer diagnosis and treatment. Although they have great biocompatibility owing to biodegradable chemical structure and low immunogenicity, sufficient information on in vivo toxicity to understand the potential risks depending on the repeated high-dose have not been adequately studied. Herein, we report the results of in vivo toxicity evaluation for CNPs focused on the number and dose of administration in healthy mice to provide a toxicological guideline for a better clinical application of CNPs. RESULTS: The CNPs were prepared by conjugating hydrophilic glycol chitosan with hydrophobic 5ß-cholanic acid and the amphiphilic glycol chitosan-5ß-cholanic acid formed self-assembled nanoparticles with its concentration-dependent homogeneous size distributions (265.36-288.3 nm) in aqueous condition. In cell cultured system, they showed significantly high cellular uptake in breast cancer cells (4T1) and cardiomyocytes (H9C2) than in fibroblasts (L929) and macrophages (Raw264.7) in a dose- and time-dependent manners, resulting in severe necrotic cell death in H9C2 at a clinically relevant highly concentrated condition. In particular, when the high-dose (90 mg/kg) of CNPs were intravenously injected into the healthy mice, considerable amount was non-specifically accumulated in major organs (liver, lung, spleen, kidney and heart) after 6 h of injection and sustainably retained for 72 h. Finally, repeated high-dose of CNPs (90 mg/kg, three times) induced severe cardiotoxicity accompanying inflammatory responses, tissue damages, fibrotic changes and organ dysfunction. CONCLUSIONS: This study demonstrates that repeated high-dose CNPs induce severe cardiotoxicity in vivo. Through the series of toxicological assessments in the healthy mice, this study provides a toxicological guideline that may expedite the application of CNPs in the clinical settings.

Smart Responsive Quercetin-Conjugated Glycol Chitosan Prodrug Micelles for Treatment of Inflammatory Bowel Diseases.

The incidence and progression of inflammatory bowel disease are closely related to oxidative stress caused by excessive production of reactive oxygen species (ROS). To develop an efficacious and safe nanotherapy against inflammatory bowel diseases (IBD), we designed a novel pH/ROS dual-responsive prodrug micelle GC-B-Que as an inflammatory-targeted drug, which was comprised by active quercetin (Que) covalently linked to biocompatible glycol chitosan (GC) by aryl boronic ester as a responsive linker. The optimized micelles exhibited well-controlled physiochemical properties and stability in a physiological environment. Time-dependent NMR spectra traced the changes in the polymer structure in the presence of H2O2, confirming the release of the drug. The in vitro drug release studies indicated a low release rate (<20 wt %) in physiological conditions, but nearly complete release (>95 wt % after 72 h incubation) in a pH 5.8 medium containing 10 µM H2O2, exhibiting a pH/ROS dual-responsive property and sustained release behavior. Importantly, the negligible drug release in a simulated gastric environment in 1 h allowed us to perform intragastric administration, which has potential to achieve the oral delivery by mature enteric-coating modification in future. Further in vivo activities and biodistribution experiments found that the GC-B-Que micelles tended to accumulate in intestinal inflammation sites and showed better therapeutic efficacy than the free drugs (quercetin and mesalazine) in a colitis mice model. Typical inflammatory cytokines including TNF-α, IL-6, and iNOS were significantly suppressed by GC-B-Que micelle treatment. Our work promoted inflammatory-targeted delivery and intestinal drug accumulation for active single drug quercetin and improved the therapeutic effect of IBD. The current study also provided an alternative strategy for designing a smart responsive nanocarrier for a catechol-based drug to better achieve the target drug delivery.

ß-Cyclodextrin/Triclosan Complex-Grafted Methacrylated Glycol Chitosan Hydorgel by Photocrosslinking via Visible Light Irradiation for a Tissue Bio-Adhesive.

Accelerating wound healing with minimized bacterial infection has become a topic of interest in the development of the new generation of tissue bio-adhesives. In this study, we fabricated a hydrogel system (MGC-g-CD-ic-TCS) consisting of triclosan (TCS)-complexed beta-cyclodextrin (ß-CD)-conjugated methacrylated glycol chitosan (MGC) as an antibacterial tissue adhesive. Proton nuclear magnetic resonance (1H NMR) and differential scanning calorimetry (DSC) results showed the inclusion complex formation between MGC-g-CD and TCS. The increase of storage modulus (G') of MGC-g-CD-ic-TCS after visible light irradiation for 200 s indicated its hydrogelation. The swollen hydrogel in aqueous solution resulted in two release behaviors of an initial burst and sustained release. Importantly, in vitro and in vivo results indicated that MGC-g-CD-ic-TCS inhibited bacterial infection and improved wound healing, suggesting its high potential application as an antibacterial tissue bio-adhesive.

Thiol-Responsive Gold Nanodot Swarm with Glycol Chitosan for Photothermal Cancer Therapy.

Photothermal therapy (PTT) is one of the most promising cancer treatment methods because hyperthermal effects and immunogenic cell death via PTT are destructive to cancer. However, PTT requires photoabsorbers that absorb near-infrared (NIR) light with deeper penetration depth in the body and effectively convert light into heat. Gold nanoparticles have various unique properties which are suitable for photoabsorbers, e.g., controllable optical properties and easy surface modification. We developed gold nanodot swarms (AuNSw) by creating small gold nanoparticles (sGNPs) in the presence of hydrophobically-modified glycol chitosan. The sGNPs assembled with each other through their interaction with amine groups of glycol chitosan. AuNSw absorbed 808-nm laser and increased temperature to 55 °C. In contrast, AuNSw lost its particle structure upon exposure to thiolated molecules and did not convert NIR light into heat. In vitro studies demonstrated the photothermal effect and immunogenic cell death after PTT with AuNSW. After intratumoral injection of AuNSw with laser irradiation, tumor growth of xenograft mouse models was depressed. We found hyperthermal damage and immunogenic cell death in tumor tissues through histological and biochemical analyses. Thiol-responsive AuNSw showed feasibility for PTT, with advanced functionality in the tumor microenvironment.

Flash Fabrication of Orally Targeted Nanocomplexes for Improved Transport of Salmon Calcitonin across the Intestine.

Salmon calcitonin (sCT) is a potent calcium-regulating peptide hormone and widely applied for the treatment of some bone diseases clinically. However, the therapeutic usefulness of sCT is hindered by the frequent injection required, owing to its short plasma half-life and therapeutic need for a high dose. Oral delivery is a popular modality of administration for patients because of its convenience to self-administration and high patient compliance, while orally administered sCT remains a great challenge currently due to the existence of multiple barriers in the gastrointestinal (GI) tract. Here, we introduced an orally targeted delivery system to increase the transport of sCT across the intestine through both the paracellular permeation route and the bile acid pathway. In this system, sCT-based glycol chitosan-taurocholic acid conjugate (GC-T)/dextran sulfate (DS) ternary nanocomplexes (NC-T) were produced by a flash nanocomplexation (FNC) process in a kinetically controlled mode. The optimized NC-T exhibited well-controlled properties with a uniform and sub-60 nm hydrodynamic diameter, high batch-to-batch reproducibility, good physical or chemical stability, as well as sustained drug release behaviors. The studies revealed that NC-T could effectively improve the intestinal uptake and permeability, owing to its surface functionalization with the taurocholic acid ligand. In the rat model, orally administered NC-T showed an obvious hypocalcemia effect and a relative oral bioavailability of 10.9%. An in vivo assay also demonstrated that NC-T induced no observable side effect after long-term oral administration. As a result, the orally targeted nanocomplex might be a promising candidate for improving the oral transport of therapeutic peptides.

Nanogels of a Succinylated Glycol Chitosan-Succinyl Prednisolone Conjugate: Release Behavior, Gastrointestinal Distribution, and Systemic Absorption.

Recently, the potential of nanoparticles (NPs) in ulcerative colitis (UC) therapy has been increasingly demonstrated. Namely, anionic NPs have been found to be accumulated efficiently to the UC damaged area due to epithelial enhanced permeability and retention (eEPR) effect. Previously, a novel anionic nanogel system (NG(S)) was prepared, and evaluated for the efficacy and toxicity. In the present study, release behaviors and biodistribution were investigated in detail to elucidate the functional mechanisms. Rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis (UC) were used as biomodels. In vitro release was examined with or without the contents of the cecum or distal colon. Gastrointestinal distribution and plasma concentrations were investigated after the intragastric administration of 10 mg prednisolone (PD) eq./kg. At pH 1.2 and 6.8, release behaviors were slow, but controlled. Overall release was not markedly different irrespective of coexistence of intestinal contents. In in vivo studies, a large amount of PD was distributed in the lower parts of the gastrointestinal tract 6 and 12 h after administration with NG(S). PD accumulated well in the colonic parts, and prolonged release was noted. The systemic absorption of PD with NG(S) was hardly found. NG(S) concentrated the drug in the colon and showed controlled release. These behaviors were considered to lead to the previously reported good results, promotion of effectiveness and suppression of toxic side effects.

Glycol chitosan-coated near-infrared photosensitizer-encapsulated gold nanocages for glioblastoma phototherapy.

Photodynamic therapy is a clinically approved treatment approach for cancer. However, it has limited applications owing to poor water solubility and the short wavelength absorption of the photosensitizer (PS). We selected a near-infrared photosensitizer, SiNC, and encapsulated into a gold nanocage (AuNC) in the presence of phase-changing material. Then, the PS-encapsulated nanocage was coated with glycol chitosan (GC) with a cleavable peptide linkage or stable cysteine linkage to protect the PS from premature release and to improve the biocompatibility of the nanocage. We obtained particles of GC-coated SiNC-encapsulated AuNC with a neutral surface charge and approximately 160 nm in size. The enzyme-cleavable peptide-linked GC formulation (GC-pep@SiNC-AuNC) showed stronger phototoxicity and tumor suppression efficacy in a glioblastoma model compared with free NIR-PS and stable cysteine-linked GC-AuNC (GC-cys@SiNC-AuNC). This polymer-coated SiNC-AuNC may be a promising agent for brain cancer phototherapy.

Glycol Chitosan-Docosahexaenoic Acid Liposomes for Drug Delivery: Synergistic Effect of Doxorubicin-Rapamycin in Drug-Resistant Breast Cancer.

Marine ecosystems are the most prevalent ecosystems on the planet, providing a diversity of living organisms and resources. The development of nanotechnology may provide solutions for utilizing these thousands of potential compounds as marine pharmaceuticals. Here, we designed a liposomal glycol chitosan formulation to load both doxorubicin (DOX) and rapamycin (RAPA), and then evaluated its therapeutic potential in a prepared drug-resistant cell model. We explored the stability of the drug delivery system by changing the physiological conditions and characterized its physicochemical properties. The electrostatic complexation between DOX-glycol chitosan and docosahexaenoic acid RAPA-liposomes (GC-DOX/RAPA ω-liposomes) was precisely regulated, resulting in particle size of 131.3 nm and zeta potential of -14.5 mV. The well-characterized structure of GC-DOX/RAPA ω-liposomes led to high loading efficiencies of 4.1% for DOX and 6.2% for RAPA. Also, GC-DOX/RAPA ω-liposomes exhibited high colloidal stability under physiological conditions and synergistic anti-cancer effects on DOX-resistant MDA-MB-231 cells, while showing pH-sensitive drug release behavior. Our results provided a viable example of marine pharmaceuticals with therapeutic potential for treating drug-resistant tumors using an efficient and safe drug delivery system.

Photo-Cured Glycol Chitosan Hydrogel for Ovarian Cancer Drug Delivery.

In this study, we prepared an injectable drug delivery depot system based on a visible light-cured glycol chitosan (GC) hydrogel containing paclitaxel (PTX)-complexed beta-cyclodextrin (ß-CD) (GC/CD/PTX) for ovarian cancer (OC) therapy using a tumor-bearing mouse model. The hydrogel depot system had a 23.8 Pa of storage modulus at 100 rad/s after visible light irradiation for 10 s. In addition, GC was swollen as a function of time. However, GC had no degradation with the time change. Eventually, the swollen GC matrix affected the releases of PTX and CD/PTX. GC/PTX and GC/CD/PTX exhibited a controlled release of PTX for 7 days. In addition, GC/CD/PTX had a rapid PTX release for 7 days due to improved water solubility of PTX through CD/PTX complex. In vitro cell viability tests showed that GC/CD/PTX had a lower cell viability percentage than the free PTX solution and GC/PTX. Additionally, GC/CD/PTX resulted in a superior antitumor effect against OC. Consequently, we suggest that the GC/CD system might have clinical potential for OC therapy by improving the water solubility of PTX, as PTX is included into the cavity of ß-CD.

Hydrogel-Mediated DOX⋠HCl/PTX Delivery System for Breast Cancer Therapy.

We used a hydrogel-mediated dual drug delivery approach, based on an injectable glycol chitosan (GC) hydrogel, doxorubicin hydrochloride (DOX⋅HCl), and a complex of beta-cyclodextrin (ß-CD) and paclitaxel (PTX) (GDCP) for breast cancer therapy in vitro and in vivo. The hydrogel was swollen over 3 days and remained so thereafter. After an initial burst period of 7 hours, the two drugs were released in a sustained manner for 7 days. The in vitro cell viability test showed that GDCP had a better anticancer effect than well plate and DOX⋅HCl/PTX (DP). In addition, the in vivo tests, which evaluated the anticancer effect, systemic toxicity, and histology, proved the feasibility of GDCP as a clinical therapy for breast cancer.

5ß-Cholanic Acid/Glycol Chitosan Self-Assembled Nanoparticles (5ß-CHA/GC-NPs) for Enhancing the Absorption of FDs and Insulin by Rat Intestinal Membranes.

The absorption-enhancing effects of glycol chitosan modified by 5ß-cholanic acid nanoparticles (5ß-CHA/GC-NPs) on a drug with poor absorption in the intestine were studied by the method of in situ closed loop. We chose fluorescein isothiocyanate-labeled dextrans (FDs) and insulin as the model drugs. 5ß-CHA/GC-NPs loaded to different drugs were prepared by the dialysis method, and the physicochemical characteristics and in vitro release profiles of nanoparticles were also estimated. The results showed that 5ß-CHA/GC-NPs markedly increased the absorption of insulin and FDs in the jejunum, ileum, and colon. The ratios of absorption for all the drugs in the jejunum were higher than those in the ileum and colon. In addition, the enhancing effect of 5ß-CHA/GC-NPs for the absorption of FDs from the jejunum was decreased with increasing molecular weights. In the toxicity test, 5ß-CHA/GC-NPs did not significantly increase the release of protein and the activities of LDH, indicating that the nanoparticles did not cause any membrane damage to the intestine. These findings suggested that 5ß-CHA/GC-NPs were safe and useful carriers for enhancing the absorption of the drug with poor absorption by intestinal membranes.

The Cocktail Effect of BMP-2 and TGF-ß1 Loaded in Visible Light-Cured Glycol Chitosan Hydrogels for the Enhancement of Bone Formation in a Rat Tibial Defect Model.

Bone tissue engineering scaffolds offer the merits of minimal invasion as well as localized and controlled biomolecule release to targeted sites. In this study, we prepared injectable hydrogel systems based on visible light-cured glycol chitosan (GC) hydrogels containing bone morphogenetic protein-2 (BMP-2) and/or transforming growth factor-beta1 (TGF-ß1) as scaffolds for bone formation in vitro and in vivo. The hydrogels were characterized by storage modulus, scanning electron microscopy (SEM) and swelling ratio analyses. The developed hydrogel systems showed controlled releases of growth factors in a sustained manner for 30 days. In vitro and in vivo studies revealed that growth factor-loaded GC hydrogels have no cytotoxicity against MC3T3-E1 osteoblast cell line, improved mRNA expressions of alkaline phosphatase (ALP), type I collagen (COL 1) and osteocalcin (OCN), and increased bone volume (BV) and bone mineral density (BMD) in tibia defect sites. Moreover, GC hydrogel containing BMP-2 (10 ng) and TGF-ß1 (10 ng) (GC/BMP-2/TGF-ß1-10 ng) showed greater bone formation abilities than that containing BMP-2 (5 ng) and TGF-ß1 (5 ng) (GC/BMP-2/TGF-ß1-5 ng) in vitro and in vivo. Consequently, the injectable GC/BMP-2/TGF-ß1-10 ng hydrogel may have clinical potential for dental or orthopedic applications.

Polymeric Micelles Based on Modified Glycol Chitosan for Paclitaxel Delivery: Preparation, Characterization and Evaluation.

Amphiphilic polymer of α-tocopherol succinate modified glycol chitosan (TS-GC) was successfully constructed by conjugating α-tocopherol succinate to the skeleton of glycol chitosan and characterized by Fourier-transform infrared (FT-IR) and proton nuclear magnetic resonance (¹H-NMR). In aqueous milieu, the conjugates self-assembled to micelles with the critical aggregation concentration of 7.2 × 10−3 mg/mL. Transmission electron microscope (TEM) observation and dynamic light scattering (DLS) measurements were carried out to determine the physicochemical properties of the micelles. The results revealed that paclitaxel (PTX)-loaded TS-GC micelles were spherical in shape. Moreover, the PTX-loaded micelles showed increased particle sizes (35 nm vs. 142 nm) and a little reduced zeta potential (+19 mV vs. +16 mV) compared with blank micelles. The X-ray diffraction (XRD) spectra demonstrated that PTX existed inside the micelles in amorphous or molecular state. In vitro and in vivo tests showed that the PTX-loaded TS-GC micelles had advantages over the Cremophor EL-based formulation in terms of low toxicity level and increased dose, which suggested the potential of the polymer as carriers for PTX to improve their delivery properties.

Glycol chitosan: A stabilizer of lipid rafts in the intestinal brush border.

Chitosan is a polycationic polysaccharide consisting of ß-(1-4)-linked glucosamine units and due to its mucoadhesive properties, chemical derivatives of chitosan are potential candidates as enhancers for transmucosal drug delivery. Recently, glycol chitosan (GC), a soluble derivative of chitosan, was shown to bind specifically to lipid raft domains in model bilayers. The small intestinal brush border membrane has a unique lipid raft composition with high amounts of glycolipids cross-linked by lectins, and the aim of the present work therefore was to study the interaction of FITC-conjugated GC (FITC-GC) with the small intestinal epithelium. Using organ culture of pig jejunal mucosal explants as a model system, we observed widespread binding of luminal FITC-GC to the brush border. Only little uptake via constitutive endocytosis into apical early endosomes occurred, unless endocytosis was induced by the simultaneous presence of cholera toxin B subunit (CTB). Biochemically, GC bound to microvillus membrane vesicles and caused a change in the density profile of detergent resistant membranes (DRMs). Collectively, the results showed that FITC-GC binds passively to lipid raft domains in the brush border, i.e. without inducing endocytosis like CTB. Instead, and unlike CTB, FITC-GC seems to exert a stabilizing, detergent-protective effect on the lipid raft organization of the brush border.

Response of Ustilago maydis against the Stress Caused by Three Polycationic Chitin Derivatives.

Chitosan is a stressing molecule that affects the cells walls and plasma membrane of fungi. For chitosan derivatives, the action mode is not clear. In this work, we used the yeast Ustilago maydis to study the effects of these molecules on the plasma membrane, focusing on physiologic and stress responses to chitosan (CH), oligochitosan (OCH), and glycol-chitosan (GCH). Yeasts were cultured with each of these molecules at 1 mg·mL-1 in minimal medium. To compare plasma membrane damage, cells were cultivated in isosmolar medium. Membrane potential (Δψ) as well as oxidative stress were measured. Changes in the total plasma membrane phospholipid and protein profiles were analyzed using standard methods, and fluorescence-stained mitochondria were observed. High osmolarity did not protect against CH inhibition and neither affected membrane potential. The OCH did produce higher oxidative stress. The effects of these molecules were evidenced by modifications in the plasma membrane protein profile. Also, mitochondrial damage was evident for CH and OCH, while GCH resulted in thicker cells with fewer mitochondria and higher glycogen accumulation.

Versatile Chemical Derivatizations to Design Glycol Chitosan-Based Drug Carriers.

Glycol chitosan (GC) and its derivatives have been extensively investigated as safe and effective drug delivery carriers because of their unique physiochemical and biological properties. The reactive functional groups such as the amine and hydroxyl groups on the GC backbone allow for easy chemical modification with various chemical compounds (e.g., hydrophobic molecules, crosslinkers, and acid-sensitive and labile molecules), and the versatility in chemical modifications enables production of a wide range of GC-based drug carriers. This review summarizes the versatile chemical modification methods that can be used to design GC-based drug carriers and describes their recent applications in disease therapy.

Visible Light-Cured Glycol Chitosan Hydrogel Containing a Beta-Cyclodextrin-Curcumin Inclusion Complex Improves Wound Healing In Vivo.

Scarless wound healing is ideal for patients suffering from soft tissue defects. In this study, we prepared a novel wet dressing (ß-CD-ic-CUR/GC) based on the visible light-cured glycol chitosan (GC) hydrogel and inclusion complex between beta-cyclodextrin (ß-CD) and curcumin (CUR). We also evaluated its efficacy in the acceleration of wound healing as compared to that of CUR-loaded GC (CUR/GC). The conjugation of glycidyl methacrylate (GM) to GC for photo-curing was confirmed by ¹H-NMR measurement, and the photo-cured GC hydrogel was characterized by the analyses of rheology, swelling ratio, SEM and degradation rate. After visible light irradiation, the surface/cross-sectional morphologies and storage (G')/loss (G'') moduli revealed the formation of hydrogel with interconnected porosity. The dressing ß-CD-ic-CUR/GC exhibited a controlled release of 90% CUR in a sustained manner for 30 days. On the other hand, CUR/GC showed CUR release of 16%. ß-CD acted as an excipient in improving the water-solubility of CUR and affected the release behavior of CUR. The in vivo animal tests including measurement of the remaining unhealed wound area and histological analyses showed that ß-CD-ic-CUR/GC may have potential as a wet dressing agent to enhance soft tissue recovery in open fractures.

siRNA Inhibition of Endocytic Pathways to Characterize the Cellular Uptake Mechanisms of Folate-Functionalized Glycol Chitosan Nanogels.

Glycol chitosan nanogels have been widely used in gene, drug, and contrast agent delivery in an effort to improve disease diagnosis and treatment. Herein, we evaluate the internalization mechanisms and intracellular fate of previously described glycol chitosan nanogels decorated with folate to target the folate receptor. Uptake of the folate-decorated nanogel was impaired by free folate, suggesting competitive inhibition and shared internalization mechanisms via the folate receptor. Nanogel uptake was shown to occur mainly through flotillin-1 and Cdc42-dependent endocytosis. This was determined by inhibition of uptake reduction observed upon siRNA depletion of these two proteins and the pathways that they regulate. The data also suggest the involvement of the actin cytoskeleton in nanogel uptake via macropinocytosis. After 7 h of incubation with HeLa cells, approximately half of the nanogel population was localized in endolysosomal compartments, whereas the remaining 50% of the material was in undefined regions of the cytoplasm. Glycol chitosan nanogels may thus have potential as drug delivery vectors for targeting different intracellular compartments.