Injectable Micelle-Incorporated Hydrogels for the Localized Chemo-Immunotherapy of Breast Tumors.

Although immune checkpoint blockade (ICB) holds potential for the treatment of various tumors, a considerable proportion of patients show a limited response to ICB therapy due to the low immunogenicity of a variety of tumors. It has been shown that some chemotherapeutics can turn low-immunogenic tumors into immunogenic phenotypes by inducing a cascade of immune responses. In this paper, we synthesized an injectable micelle-incorporated hydrogel, which was able to sequentially release the chemotherapeutic gemcitabine (GEM) and the hydrophobic indoleamine 2, 3-dioxygenase inhibitor, d-1-methyltryptophan (d-1MT) at tumor sites. The hydrogel was formed via the thiol-ene click reaction between the thiolated chondroitin sulfate and the micelle formed by amphiphilic methacrylated Pluronic F127, in which hydrophobic d-1MT was encapsulated in the core of the F127 micelles and the hydrophilic GEM was dispersed in the hydrogel network. The successive release of chemotherapeutics and immune checkpoint inhibitors at tumor tissues will first promote the infiltration of cytotoxic T lymphocytes and subsequently induce a robust antitumor immune response, ultimately exerting a synergetic therapeutic efficacy. In a 4T1 tumor-bearing mice model, our results showed that the combination of chemotherapy and immunotherapy through the micelle-incorporated hydrogel triggered an effective antitumor immune response and inhibited tumor metastasis to the lung. Our results highlight the potential of the injectable micelle-incorporated hydrogel for the localized chemo-immunotherapy in the treatment of breast tumors.

The synthesis and biological evaluation of chondroitin sulfate E glycodendrimers.

Aim: Chondroitin sulfate (CS) is a class of highly sulfated polysaccharides that possess many important biological functions. The heterogeneity of CS limits pharmacological research and leads to ambiguous mechanisms. Thus, glycomimetics are demanded as replacement of natural polysaccharides to explore important biological processes. Results & methodology: Here the preparation of CS glycodendrimers is reported as well as their use as CS mimetics to regulate the NF-κB pathway. Multivalent presentation of sugar epitopes on appropriate dendrimer scaffolds increased the suppression of the NF-κB pathway. The interaction between CS-E molecules and TNF-α was examined by nuclear magnetic resonance technology. Conclusion: Overall, the glycodendrimer reported here may be potentially employed as molecular tool to investigate the biological functions of CS.

Controlling methacryloyl substitution of chondroitin sulfate: injectable hydrogels with tunable long-term drug release profiles.

Drug delivery systems capable of local sustained release of small molecule therapeutics remain a critical need in many fields, including oncology. Here, a system to create tunable hydrogels capable of modulating the loading and release of cationic small molecule therapeutics was developed. Chondroitin sulfate (CS) is a sulfated glycosaminoglycan that has many promising properties, including biocompatibility, biodegradation and chemically modifiable groups for both covalent and non-covalent bonding. CS was covalently modified with photocrosslinkable methacryloyl groups (CSMA) to develop an injectable hydrogel fabrication. Utilizing anionic groups, cationic drugs can be adsorbed and released from the hydrogels. This study demonstrates the synthesis of CSMA with a varying degree of substitution (DS) to generate hydrogels with varying swelling properties, maximum injection force, and drug release kinetics. The DS of the synthesized CSMA ranged from 0.05 ± 0.02 (2 h reaction) to 0.28 ± 0.02 (24 h reaction) with a DS of 1 representing 100% modification. The altered DS resulted in changes in hydrogel properties with the swelling of 20% CSMA hydrogels ranging from 42 (2 h reaction) to 13 (24 h reaction) and injection forces ranging from 18 N (2 h reaction) to 94 N (24 h reaction). The release of sunitinib, an oncology therapeutic that inhibits intracellular signaling by targeting multiple receptor tyrosine kinases, ranged from 18 µg per day (2 h reaction) to 9 µg per day (24 h reaction). While decreasing the DS increased the hydrogel swelling and rate of therapeutic release, it also limited the hydrogel fabrication range to only those containing 10% or higher CSMA. Blended polymer systems with poly(vinyl alcohol)-methacrylate (PVAMA) were fabricated to stabilize the resulting hydrogels via attenuating the swelling properties. Release profiles previously unattainable with the pure CSMA hydrogels were achieved with the blended hydrogel formulations. Overall, these studies identify a method to formulate tunable CSMA and blended CSMA/PVAMA hydrogels capable of sustained release of cationic therapeutics over six weeks with applications in oncology therapeutics.

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Preparation and in vitro evaluation of novel cross-linked chondroitin sulphate nanoparticles by aluminium ions for encapsulation of green tea flavonoids.

Chondroitin sulphate is a sulphated glycosaminoglycan biopolymer composed over 100 individual sugars. Chondroitin sulphate nanoparticles (NPs) loaded with catechin were prepared by an ionic gelation method using AlCl3 and optimised for polymer and cross-linking agent concentration, curing time and stirring speed. Zeta potential, particle size, loading efficiency, and release efficiency over 24 h (RE24%) were evaluated. The surface morphology of NPs was investigated by scanning electron microscopy and their thermal behaviour by differential scanning calorimetric. Antioxidant effect of NPs was determined by chelating activity of iron ions. The cell viability of mesenchymal stem cells was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide assay and the calcification of osteoblasts was studied by Alizarin red staining. The optimised NPs showed particle size of 176 nm, zeta potential of -20.8 mV, loading efficiency of 93.3% and RE24% of 80.6%. The chatechin loaded chondroitin sulphate NPs showed 70-fold more antioxidant activity, 3-fold proliferation effect and higher calcium precipitation in osteoblasts than free catechin.

Synthesis of Fucosylated Chondroitin Sulfate Glycoclusters: A Robust Route to New Anticoagulant Agents.

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan with excellent anticoagulant activity. Studies show that FuCS and its depolymerized fragments exhibit a different anticoagulant mechanism from that of heparin derivatives, with decreased risks of adverse effects and bleeding. However, further exploitation has been hindered by the scarcity of structurally defined oligosaccharides. Herein, facile method is reported for the synthesis of the repeating trisaccharide unit of FuCS based on the degradation of chondroitin sulfate polymers. A series of simplified FuCS glycomimetics that have highly tunable structures, controllable branches, and defined sulfation motifs were generated by copper-catalyzed alkyne-azide cycloaddition. Remarkable improvement in activated partial thromboplastin time (APTT) assay activities was observed as the branches increased, but no significant influences were observed for prothrombin time (PT) and thrombin time (TT) assay activities. Further FXase inhibition tests suggested that glycoclusters 33 b-40 b selectively inhibited intrinsic anticoagulant activities, but had little effect on the extrinsic and common coagulation pathways. Notably, glycoclusters with the 2,4-di-O-sulfated fucosyl residue displayed the most potency, which was in consistent with that of natural polysaccharides. These FuCS clusters demonstrated potency to mimic linear glycosaminoglycans and offer a new framework for the development of novel anticoagulant agents.

Fucosylated chondroitin sulfate oligosaccharides exert anticoagulant activity by targeting at intrinsic tenase complex with low FXII activation: Importance of sulfation pattern and molecular size.

Fucosylated chondroitin sulfates (fCSs) are structurally unusual glycosaminoglycans isolated from sea cucumbers that exhibit potent anticoagulant activity. These fCSs were isolated from sea cucumber, Isostichopus badionotus and Pearsonothuria graeffei. Fenton reaction followed by gel filtration chromatography afforded fCS oligosaccharides, with different sulfation patterns identified by mass and NMR spectroscopy, and these were used to clarify the relationship between the structures and the anticoagulant activities of fCSs. In vitro activities were measured by activated partial thromboplastin time (APTT), thrombin time (TT), thrombin and factor Xa inhibition, and activation of FXII. The results showed that free radicals preferentially acted on GlcA residues affording oligosaccharides that were purified from both fCSs. The inhibition of thrombin and factor X activities, mediated through antithrombin III and heparin cofactor II of fCSs oligosaccharides were affected by their molecular weight and fucose branches. Oligosaccharides with different sulfation patterns of the fucose branching had a similar ability to inhibit the FXa by the intrinsic factor Xase (factor IXa-VIIIa complex). Oligosaccharides with 2,4-O-sulfo fucose branches from fCS-Ib showed higher activities than ones with 3,4-O-disulfo branches obtained from fCS-Pg. Furthermore, a heptasaccharide is the minimum size oligosaccharide required for anticoagulation and FXII activation. This activity was absent for fCS oligosaccharides smaller than nonasaccharides. Molecular size and fucose branch sulfation are important for anticoagulant activity and reduction of size can reverse the activation of FXII caused by native fCSs.

Synthesis of strontium chondroitin sulfate and the evaluation of its capability to attenuate osteoarthritis.

Osteoarthritis (OA) is the most prevalent musculoskeletal disorder and the leading cause of joint disability in elderly patients. In this study, we fabricated strontium chondroitin sulfate (SrCS), a new polysaccharide-metal ion complex that is the combination of chondroitin sulfate and strontium, which are two widely adopted chemicals in OA clinical management. The structural, chemical compositions and morphology of as-fabricated SrCS were systematically investigated. Cell proliferation test, RT-PCR and preliminary animal studies were conducted to evaluate the clinical potential of SrCS on OA treatment. The materials characterization results verified that the Sr was successfully integrated into CS by replacing sodium in the original structure and formed a new polysaccharide-metal ion complex. The cell proliferation results indicated that the SrCS has excellent biocompatibility for both chondrocyte and osteoblast. The RT-PCR results showed that the SrCS can significantly increase the expression of COLII and ACAN, decrease MMP1 and MMP13 in chondrocyte and decrease the IL-6 and IL-1ß in both chondrocyte and osteoblast. Preliminary animal studies demonstrated that SrCS can effectively simulate the articular cartilage formation in SD-rats after modified Hulth's OA modeling surgery. We therefore believed that the SrCS should be a rather effective chemical for OA clinical management as well as a beneficial component for various biomaterials in cartilage tissue engineering.

Interpreting the behavior of concentration-response curves of hyaluronidase inhibitors under DMSO-perturbed assay conditions.

Hyaluronan-degrading enzyme (hyaluronidase) is involved in tumor growth and inflammation, and as such, hyaluronidase inhibitors have received recent attention as potential therapeutics. The previous studies have successfully discovered a wide range of inhibitors, but unfortunately most of them are dissimilar to original ligand hyaluronan and the mode of action is poorly understood. The present study mechanistically characterized these structurally unrelated inhibitors by interpreting the behavior of concentration-response curves under several in vitro assay conditions. Detergent-addition conditions definitely identified aggregation-based inhibitors. Subsequently, DMSO-perturbed conditions, though preliminary, highlighted the inhibitors that might bind to enzyme non-specifically. Here, an intriguing implication of the latter description is that DMSO-perturbed conditions would generate non-productive but not-denatured enzyme that is an assembly of effective species to capture non-specific binding molecules, and thereby would attenuate their inhibitory activities.

Divergent Synthesis of Chondroitin Sulfate Disaccharides and Identification of Sulfate Motifs that Inhibit Triple Negative Breast Cancer.

Glycosaminoglycans (GAGs) regulate many important physiological processes. A pertinent issue to address is whether GAGs encode important functional information via introduction of position specific sulfate groups in the GAG structure. However, procurement of pure, homogenous GAG motifs to probe the "sulfation code" is a challenging task due to isolation difficulty and structural complexity. To this end, we devised a versatile synthetic strategy to obtain all the 16 theoretically possible sulfation patterns in the chondroitin sulfate (CS) repeating unit; these include rare but potentially important sulfated motifs which have not been isolated earlier. Biological evaluation indicated that CS sulfation patterns had differing effects for different breast cancer cell types, and the greatest inhibitory effect was observed for the most aggressive, triple negative breast cancer cell line MDA-MB-231.

Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase.

Fucosylated chondroitin sulfate (FCS), a structurally unusual glycosaminoglycan, has distinct anticoagulant properties, and is an especially strong inhibitor of the intrinsic factor Xase (anti-Xase). To obtain a highly selective inhibitor of human Xase, we purified six native FCSs with various sulfation patterns, prepared a series of FCS derivatives, and then elucidated the relationship between the structures and the anticoagulant activities of FCSs. FCSs 1-3 containing higher Fuc2S4S exhibit stronger AT-dependent anti-IIa activities, whereas 4-6 containing more Fuc3S4S produce potent HCII-dependent anti-IIa activities. Saccharides containing a minimum of 6-8 trisaccharide units, free carboxyl groups, and full fucosylation of GlcA may be required for potent anti-Xase activity, and approximately six trisaccharide units and partial fucosylation of GlcA may contribute to potent HCII-dependent activity. Decreasing of the molecular weights markedly reduces their AT-dependent anti-IIa activities, and even eliminates human platelet and factor XII activation. Furthermore, in vitro and in vivo studies suggested that fractions of 6-12 kDa may be very promising compounds as putative selective intrinsic Xase inhibitors with antithrombotic action, but without the consequences of major bleeding and factor XII activation.

Facile preparation of pH-sensitive micelles self-assembled from amphiphilic chondroitin sulfate-histamine conjugate for triggered intracellular drug release.

Stimuli-responsive materials, enabling drugs to be released in the acidic tumor and intracellular microenvironments, draw an increasing attention in chemotherapy. Here novel pH-sensitive biodegradable micelles are fabricated using a one-step, one-medium process without organic solvent for efficient loading and rapid intracellular release of hydrophobic cargos. The amphiphilic chondroitin sulfate-histamine conjugate (CS-his) were successfully synthesized and assembled into nanoparticles in aqueous medium with desirable size (133 nm) and low critical micelle concentration (CMC) (0.05 mg/L). Owning to the pH-sensitive structure of imidazole, the nanoparticles show pH-responsive behavior upon reducing the pH value of surrounding media, accompany with formation of large aggregates and increase of ζ potential. When the nanoparticles were utilized to deliver the model drug DOX, they exhibited a specific on-off switch drug release behavior, triggering DOX release in acidic surroundings (intracellular endosomes) and sealing DOX in neutral surroundings (blood circulation or extracellular matrix). CCK-8 assays and confocal laser scanning microscopy (CLSM) against HepG2 cells indicated that the nanoparticles themselves had no associated cytotoxicity, while drug-loaded nanoparticles possessed high cytotoxicity to model cells and presented high efficiency of cellular uptake. These flexible micelles with an on-off switched drug release may offer a promising pattern to accurately deliver a wide variety of hydrophobic payloads to tumor cells for cancer therapy.

Holothurian fucosylated chondroitin sulfate.

Fucosylated chondroitin sulfate (FucCS) is a structurally distinct glycosaminoglycan found in sea cucumber species. It has the same backbone composition of alternating 4-linked glucuronic acid and 3-linked N-acetyl galactosamine residues within disaccharide repeating units as regularly found in mammalian chondroitin sulfates. However, FucCS has also sulfated fucosyl branching units 3-O-linked to the acid residues. The sulfation patterns of these branches vary accordingly with holothurian species and account for different biological actions and responses. FucCSs may exhibit anticoagulant, antithrombotic, anti-inflammatory, anticancer, antiviral, and pro-angiogenic activities, besides its beneficial effects in hemodialysis, cellular growth modulation, fibrosis and hyperglycemia. Through an historical overview, this document covers most of the science regarding the holothurian FucCS. Both structural and medical properties of this unique GAG, investigated during the last 25 years, are systematically discussed herein.

The copolymer of Poly(2-dimethylaminoethyl methacrylate) and methacrylated chondroitin sulfate with low cytotoxicity for gene delivery.

Poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) is one of the most potent synthetic nonviral gene-delivery vectors because of its high transfection efficiency. However, the cytotoxicity of PDMAEMA is a major concern for its clinical applications. An anionic crosslinker is synthesized based on a natural polysaccharide, chondroitin sulfate (CS), by introducing methacrylate groups (CSMA). By systematically adjusting the substitution degree of methacrylation on CS and the weight percent of CSMA and PDMAEMA, sol-type copolymers are obtained as a gene-delivery vector. The combination of CS and PDMAEMA is expected not only to reduce the cytotoxicity of PDMAEMA, but also to facilitate better transfection efficiency than PDMAEMA because of the recognition of CS by CD44 receptors on cell surfaces. Two CSMA-modified PDMAEMA copolymers with different CSMA constituents are selected and their polyplexes prepared with plasmid DNA. The cytotoxicity and gene transfection efficiency of the polyplexes are tested and compared with those of PDMAEMA/pDNA. The copolymers of CSMA and PDMAEMA show significantly improved cell viability as compared with PDMAEMA. Their formed polyplexes with pDNA also show lower cytotoxicity than does PDMAEMA/pDNA. The transfection efficiency remarkably increases as the CSMA-modified PDMAEMA/pDNA polyplex is prepared at a weight ratio of 2.4. The internalization mechanism of CSMA-modified PDMAEMA/pDNA in HEK 293T cells is mainly based on caveolae-mediated endocytosis. However, both caveolae-mediated and CD44-mediated endocytosis mechanisms are involved in U87 cells.

Toward libraries of biotinylated chondroitin sulfate analogues: from synthesis to in vivo studies.

Chondroitin sulfate-E (CS-E) oligosaccharidic analogues (di to hexa) were prepared from lactose. In these compounds, the 2-acetamido group was replaced by a hydroxyl group. This modification speeded up the synthesis, and large oligosaccharides were constructed in a few steps from a lactose-originated block. The protecting groups used were as follows; Fmoc for hydroxyl groups to be glycosylated, allyl group for anomeric position protection, and trichoroacetimidate leaving groups were used to prepare up to octasaccharides. We took advantage of the presence of allyl group to develop a click biotinylation, through its transformation into a 3-azido-2-hydroxyl propyl group in two steps (epoxidation and sodium azide epoxide opening). The biotinylating agent was a water-soluble propargylated and biotinylated triethylene glycol (PEG). By using surface plasmon resonance (SPR), it was shown that the di-, tetra-, and hexasaccharides display a binding affinity and selectivity toward HSF/GSF and CXCL12 similar to that of CS-E. A parallel study confirmed their mimicry of natural compounds, based on the hexasaccharide interaction with Otx2, a homeodomain protein involved in brain maturation, thus validating our simplification approach to synthesize bioactive GAG.

An orthopedic tissue adhesive for targeted delivery of intraoperative biologics.

Tissue adhesives can bind together damaged tissues and serve as tools to deliver and localize therapeutics to facilitate regeneration. One emerging therapeutic trend in orthopedics is the use of intraoperative biologics (IOB), such as bone marrow (BM) and platelet-rich plasma (PRP), to stimulate healing. Here, we introduce the application of the biomaterial chondroitin sulfate succinimidyl succinate (CS-NHS) to deliver IOB in a hydrogel adhesive. We demonstrate the biomaterial's ability to bind various tissue types and its cellular biocompatibility with encapsulated human mesenchymal stem cells (hMSCs). Further, we examine in detail the CS-NHS adhesive combined with BM aspirate for use in bone applications. hMSCs were encapsulated in CS-BM and cultured for 5 weeks in osteogenic medium. Quantitative RT-PCR demonstrated osteogenesis via upregulation of the osteogenic transcription factor Runx2 and bone markers alkaline phosphatase and osteocalcin. Significant deposition of calcium and osteocalcin was detected using biochemical, histological, and immunohistochemical techniques. Shear testing demonstrated that the CS-BM adhesive exhibited an adhesive strength approximately an order of magnitude stronger than fibrin glue and approaching that of a cyanoacrylate adhesive. These results indicate that CS-NHS is a promising delivery tool for IOB in orthopedic applications requiring a strong, degradable, and biocompatible adhesive that supports bone growth.

Chondroitin sulfate-g-poly(ϵ-caprolactone) co-polymer aggregates as potential targeting drug carriers.

The aim of this study is to delineate the effect of various amounts of hydrophobic polycaprolactone (PCL) grafted onto three different degrees of methacrylated chondroitin sulfate (CSMA) on chemical-physical properties. The co-polymers were prepared by reacting the modified PCL and the hydrophilic CSMA via a radical reaction (CSMA-PCL). The effect of degree of methacrylation of CSMA and feed ratio between CSMA and PCL on compositions and critical micelle concentrations was systematically studied. The PCL composition of the CSMA-PCL was characterized by (1)H-NMR and FT-IR. The hydrodynamic diameters and morphologies of CSMA-PCL micelles were studied by DLS and TEM. Critical micelle concentrations were determined using pyrene as a probe. Taking one of the CSMA-PCL micelles as an example, a cancer-mediated ligand, folic acid, was linked to the surface. The cellular uptake of the folic acid-linked CSMA-PCL in folate-receptor-overexpressing KB cells was studied by confocal laser scanning microscopy and flow cytometry.

Potential of self-organizing nanogel with acetylated chondroitin sulfate as an anti-cancer drug carrier.

In order to obtain feasibility data regarding the possibility of using chondroitin sulfate (CS) in an anti-cancer drug delivery system, CS was chemically modified by a one-step process with acetic anhydride. Although 3 samples with different degrees of acetylation were synthesized, only the sample with the highest degree of acetylation (AC-CS3) was tested as a nanogel because the others (AC-CS1 and 2) dissolved in distilled water (DW) in the test range (1-10 mg/ml). The AC-CS3 nanogel was characterized by fluorescence probe and dynamic light scattering (DLS) techniques. Its critical aggregation concentration (CAC) was <2.0 x 10(-2) mg/ml at 25 degrees C. The partition equilibrium constant, K(v), of the nanogel (7.88 x 10(5)) was similar to that of polymeric micelles, which means that the acetyl group may act as a hydrophobic core controlling pharmacokinetic behavior. The higher surface charge value in the nanogel, above - 40 due to carboxyl and sulfate groups in CS, explains its good stability. The anticancer drug doxorubicin (DOX) loading efficiency of the AC-CS3 nanogel was also superior, at above 90%. Changes in the size of the polydispersion index (PDI) of nanogels loaded with DOX over a 3-week period were negligible. The nanogels interacted with HeLa cells and were internalized together with the entrapped drug within the cytoplasm, probably via an endocytic mechanism exploited by sugar receptors. Based on these results, the AC-CS3 nanogel is expected to prove useful as an anti-cancer drug carrier for chemotherapy.

Novel proteoglycan glycotechnology: chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application.

Proteoglycans consist of a protein core, with one or more glycosaminoglycan chains (i.e., chondroitin sulfate, dermatan sulfate and heparin sulfate) bound covalently to it. The glycosaminoglycan chains account for many of the functions and properties of proteoglycans. The development of proteoglycan glycotechnology to exploit the functionality of glycosaminoglycan chains is an extremely important aspect of glycobiology. Here we describe an efficient and widely applicable method for chemoenzymatic synthesis of conjugate compounds comprising intact long chondroitin sulfate (ChS) chains. An alkyne containing ChS was prepared by an enzymatic transfer reaction and linked with a chemically synthesized core compound containing an azido group using click chemistry. This method enabled highly efficient introduction of ChS into target materials. Furthermore, the ChS-introduced compounds had marked stability against proteolysis, and the chemically linked ChS chain contributed to the stability of these core compounds. We believe the present method will contribute to the development of proteoglycan glycobiology and technology.

Folate-mediated chondroitin sulfate-Pluronic 127 nanogels as a drug carrier.

Pluronic F127 (PF127), one of the polymers which can inhibit drug efflux transporters in cancer therapy, was used to produce amphiphilic nanocarriers for doxorubicin (DOX). In order to stabilize the nanocarriers, the hydroxyl groups on both termini of PF127 were acrylated and reacted with methacrylated chondroitin sulfate (CSMA) to form CS-PF127 nanogel. The introducing CSMA has carboxylic acid groups which can be used to react with a folic acid-polyethylene glycol (FA-PEG). Folic acid, having high binding affinity to tumor-associated folate receptors (FR), provides a selective delivery of doxorubicin (DOX) to FR-positive tumor cells. DOX was loaded either in a cationic DOX.HCl form through the electrostatic interactions with the negative charges of chondroitin sulfate, or in a free DOX form by solubilization into the PPO core compartment of PF127. The loading efficiency and release behavior of DOX prepared from two different formulations are compared. The synthesis of CS-PF127 and FA-PEG grafted CS-PF127 (FA-CS-PF127) was characterized by nuclear magnetic resonance spectrometry (NMR), ultraviolet/visible spectroscopy (UV), and X-ray photoelectron spectroscopy (XPS). With a fluorescent probe technique, the critical aggregation concentrations (CAC) are 7.5 x 10(-2)mg/mL for CS-PF127 and 7.9 x 10(-2)mg/mL for FA-CS-PF127, respectively. The spherical images of nanogels were visualized with the use of the transmission electron microscope (TEM). The particle diameters measured by dynamic light scattering (DLS) are 299.6+/-8.2nm for CS-PF127 and 138.3+/-12.3 for FA-CS-PF127, neither aggregation nor change in sizes in double deionized (DD) water after 20 days. The better cellular uptake of FA-CS-PF127 in KB cells was evidenced by confocal laser scanning microscopy (CLSM) and flow cytometry upon loading Rhodamine123 as a probe.