Glutathione S-transferase theta 1 protects against colitis through goblet cell differentiation via interleukin-22.

The enzyme glutathione S-transferase theta 1 (GSTT1) is involved in detoxifying chemicals, including reactive oxygen species (ROS). Here, we provide a significant insight into the role of GSTT1 in inflammatory bowel disease (IBD). We identified decreased expression of GSTT1 in inflamed colons from IBD patients compared to controls. We intrarectally or intraperitoneally delivered Gstt1 gene to mice with dextran sodium sulfate (DSS)-induced colitis and noted attenuation of colitis through gene transfer of Gstt1 via an IL-22 dependent pathway. Downregulation of GSTT1 by pathogen-associated molecular patterns (PAMPs) of microbes reduced innate defense responses and goblet cell differentiation. The GSTT1 mutation in intestinal epithelial cells (IECs) and IBD patients decreased its dimerization, which was connected to insufficient phosphorylation of signal transducer and activator of transcription-3 and p38/mitogen-activated protein kinase by their common activator, IL-22. GSTT1 ameliorated colitis and contributed as a modulator of goblet cells through sensing pathogens and host immune responses. Its mutations are linked to chronic intestinal inflammation due to its insufficient dimerization. Our results provide new insights into GSTT1 mutations that are linked to chronic intestinal inflammation due to its insufficient dimerization and their functional consequences in IBDs.

Lactobacillus plantarum CBT LP3 ameliorates colitis via modulating T cells in mice.

Lactobacillus plantarum has been identified as a probiotic bacterium owing to its role in immune regulation and maintenance of intestinal permeability. Here, we investigated the anti-colitic effects and mechanism of L. plantarum CBT LP3 (LP3). This in vivo study was performed using dextran sodium sulfate (DSS) to induce colitis in mice. Mice were randomly divided into three groups: a control supplied with normal drinking water, a DSS-treated group followed by oral administration of vehicle, and a DSS-treated group gavaged with LP3 daily for 7 days following DSS administration. An analysis of macrophages and T cell subsets harvesting from peritonium cavity cells and splenocytes was performed using a flow cytometric assay. Gene expression and cytokine profiles were measured using quantitative reverse transcriptase polymerase chain reaction. The administration of LP3 significantly attenuated disease activity and histolopathology compared to control. LP3 had anti-inflammatory effects, with increased induction of regulatory T cells and type 2 helper T cells in splenocytes and restoration of goblet cells accompanied by suppression of proinflammatory cytokine expressions. These findings suggest that L. plantarum CBT LP3 can be used as a potent immunomodulator, which has significant implications for IBD treatment.

Lactobacillus acidophilus suppresses intestinal inflammation by inhibiting endoplasmic reticulum stress.

BACKGROUND AND AIM: Nuclear factor kappa B (NF-κB) activation and endoplasmic reticulum (ER) stress signaling play significant roles in the pathogenesis of inflammatory bowel disease (IBD). Thus, we evaluated whether new therapeutic probiotics have anti-colitic effects, and we investigated their mechanisms related to NF-κB and ER-stress pathways. METHODS: Luciferase, nitric oxide, and cytokine assays using HT-29 or RAW264.7 cells were conducted. Mouse colitis was induced using dextran sulfate sodium and confirmed by disease activity index and histology. Macrophages and T-cell subsets in isolated peritoneal cavity cells and splenocytes were analyzed by flow cytometry. Gene and cytokine expression profiles were determined using reverse-transcription polymerase chain reaction. RESULTS: Lactobacillus acidophilus (LA1) and Pediococcus pentosaceus inhibited nitric oxide production in RAW264.7 cells, but only LA1 inhibited Tnfa and induced Il10 expression. LA1 increased the lifespan of dextran sulfate sodium-treated mice and attenuated the severity of colitis by inducing M2 macrophages in peritoneal cavity cells and Th2 and Treg cells in splenocytes. The restoration of goblet cells in the colon was accompanied by the induction of Il10 expression and the suppression of pro-inflammatory cytokines. Additionally, we found that LA1 exerts an anti-colitic effect by improving ER stress in HT-29 cells as well as in vivo. CONCLUSIONS: We showed that LA1 significantly interferes with ER stress and suppresses NF-κB activation. Our findings suggest that LA1 can be used as a potent immunomodulator in IBD treatment, and the regulation of ER stress may have significant implications in treating IBD.

Protective effects of guggulsterone against colitis are associated with the suppression of TREM-1 and modulation of macrophages.

Triggering receptor expressed on myeloid cells 1 (TREM-1)-expressing intestinal macrophages are significantly increased in the colons of patients with inflammatory bowel disease (IBD). We focused here on the effects of guggulsterone on macrophage modulation in colitis as a potential therapeutic molecule in human IBD and explore the underlying mechanisms. Gene expression in macrophages was examined and wound-healing assay using HT-29 cells was performed. Colitis in wild-type and IL-10-, Toll-like receptor 4 (TLR4)-, and myeloid differentiation primary response 88 (MyD88)-deficient mice was induced via the administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the colon. In both in vitro and in vivo experiments, guggulsterone suppressed intestinal inflammation amplified by TREM-1 stimulation, in which the suppression of NF-κB, activating protein-1, and proteasome pathways was involved. In the TNBS-induced colitis model, guggulsterone reduced disease activity index scores and TREM-1 expression, stimulated IL-10 production, and improved survival in wild-type mice. These effects were not observed in IL-10-, TLR4-, and MyD88-deficient mice. Guggulsterone also suppressed M1 polarization, yet induced the M2 phenotype in macrophages from IBD patients as well as from mice. These findings indicate that guggulsterone blocks the hyperactivation of macrophages via TREM-1 suppression and induces M2 polarization via IL-10 mediated by the TLR4 signaling pathway. Furthermore, this study provides a new rationale for the therapeutic potential of guggulsterone in the treatment of IBD. NEW & NOTEWORTHY We found that guggulsterone attenuates triggering receptor expressed on myeloid cells 1 (TREM-1)-mediated hyperactivation of macrophages and polarizes macrophages toward the M2 phenotype. This was mediated by IL-10 and partly Toll-like receptor 4 signaling pathways. Overall, these data support that guggulsterone as a natural plant sterol modulates macrophage phenotypes in colitis, which may be of novel therapeutic importance in inflammatory bowel disease treatment.

Does tranexamic acid increase the risk of thromboembolism after bilateral simultaneous total knee arthroplasties in Asian Population?

PURPOSE: To ascertain whether tranexamic acid reduces the blood loss and transfusion rate and volumes; increase the prevalence of deep vein thrombosis (DVT); and investigate factors associated with DVT in patients undergoing primary bilateral total knee arthroplasties (TKAs) without use of chemical thromboprophylaxis. METHODS: There were 874 patients (1748 knees) in the control group who did not receive tranexamic acid and 871 patients (1742 knees) in the study group who received tranexamic acid. Mechanical compression device was applied without any chemical thromboprophylaxis. Transfusion rates and volumes were recorded. DVT was diagnosed using both sonogram and venogram at 7 or 8 day post-operatively. RESULTS: Intra- and post-operative blood loss and transfusion volumes were significantly lower in the tranexamic acid group. The prevalence of DVT was 14% (245 of 1748 knees) in the control group and 18% (314 of 1742 knees) in the tranexamic acid group. Pre- and post-operative perfusion lung scans revealed no evidence of PE in any patients in either group. Coagulation or thrombophilic data or molecular genetic testing was not significantly different between the two groups. CONCLUSION: The use of tranexamic acid reduces the volume of blood transfusion and does not increase the prevalence of DVT or PE in the patients who did not receive routine chemical thromboprophylaxis after primary bilateral simultaneous sequential TKAs in Asian patients.

Preliminary Study on an Alternative Test Method with MCTT HCETM for Ocular Irritation Test of Ophthalmic Medical Devices.

The sustained growth of the market for ophthalmic medical devices has increased the demand for alternatives to animal testing for the evaluation of eye irritation. The International Organization for Standardization has acknowledged the need to develop novel in vitro tests to replace animal testing. Here, we evaluated the applicability of an alternative method based on a human corneal model to test the safety of ophthalmic medical devices. 2-Hydroxyethyl methacrylate (HEMA) and Polymethyl methacrylate (PMMA), which are used to fabricate contact lenses, were used as base materials. These materials were blended with eye irritant and non-irritant chemicals specified in the OECD Test Guideline (TG) 492 and Globally Harmonized System (GHS) classification. Then, three GLP-certified laboratories performed three replicates using the developed method using 3D reconstructed human cornea epithelium, MCTT HCETM. OECD TG 492 describes the procedure used to evaluate the eye hazard potential of the test chemical based on its ability to induce cytotoxicity in a reconstructed human cornea-like epithelium (RhCE) tissue. Results: The within-laboratory reproducibility (WLR) and between-laboratory reproducibility (BLR) were both 100%. When a polar extraction solvent was used, the sensitivity, specificity, and accuracy were all 100% in each laboratory. When a non-polar extraction solvent was used, the sensitivity was 80%, the specificity was 100%, and the accuracy was 90%. The proposed method exhibited excellent reproducibility and predictive capacity within and between laboratories. Therefore, the proposed method using the MCTT HCETM model could be used to evaluate eye irritation caused by ophthalmic medical devices.

Olfactomedin 4 produces dysplasia but suppresses metastasis of colon cancer.

Development of colorectal cancer (CRC) is regulated by a series of genetic and microenvironmental alterations. Olfactomedin 4 (OLFM4) is a secreted glycoprotein that is highly expressed in the gastrointestinal tract and modulates inflammation. However, the role of OLFM4 in CRC is uncertain. Here we aimed to explore the function of OLFM4 in CRC in vivo and in vitro. The mRNA expression of OLFM4 was up-regulated in precursor lesions with dysplasia or ulcerative colitis but was reduced in CRC. OLFM4 neutralizing antibody suppressed inflammation-mediated early-stage CRC formation in an AOM/DSS colitis-associated cancer model. OLFM4 knockdown cells exhibited increased cell proliferation and motility in vitro and in vivo. Ablation of OLFM4 increased tumor growth and metastasis in xenograft experiments. In addition, OLFM4 knockdown cells showed elevated expression of colon cancer stem cell markers including CD133, resulting in increased metastasis via epithelial-mesenchymal transition signaling. This study demonstrated that OLFM4 regulates inflammation and cancer progression differently; ablation of OLFM4 promotes cancer metastasis via stemness and epithelial-mesenchymal transition. These results suggest a new route for controlling cancer progression and metastasis.

Pre-validation study of spectrophotometric direct peptide reactivity assay (Spectro-DPRA) as a modified in chemico skin sensitization test method.

Skin sensitization is induced when certain chemicals bind to skin proteins. Direct peptide reactivity assay (DPRA) has been adopted by the OECD as an alternative method to evaluate skin sensitization by assessing a substance's reaction to two model peptides. A modified spectrophotometric method, Spectro-DPRA, can evaluate skin sensitization, in a high throughput fashion, to obviate some limitations of DPRA. Pre-validation studies for Spectro-DPRA were conducted to determine transferability and proficiency, within- and between-laboratory reproducibility, and predictive ability based on GLP principles at three laboratories (AP, KTR, and KCL). All laboratories confirmed high (> 90%) concordance for evaluating the sensitivity induced by ten chemical substances. The concordance among the three tests performed by each laboratory was 90% for AP, 100% for KTR, and 100% for KCL. The mean accuracy of the laboratories was 93.3% [compared to the standard operating procedure (SOP)]. The reproducibility among the three laboratories was as high as 86.7%; the accuracy was 86.7% for AP, 100% for KTR, and 86.7% for KCL (compared to the SOP). An additional 54 substances were assessed in 3 separate labs to verify the prediction rate. Based on the result, 29 out of 33 substances were classified as sensitizers, and 19 out of 21 identified as non-sensitizers; the corresponding sensitivity, specificity, and accuracy values were 87.9%, 90.5%, and 88.9%, respectively. These findings indicate that the Spectro-DPRA can address the molecular initiating event with improved predictability and reproducibility, while saving time and cost compared to DPRA or ADRA.

Triggering Receptor Expressed on Myeloid Cells-1 Agonist Regulates Intestinal Inflammation via Cd177+ Neutrophils.

Triggering receptor expressed on myeloid cell-1 (TREM-1) signaling is expressed on neutrophils and monocytes that is necessary for the successful antimicrobial response and resolution of inflammation in the gut. In this study, we determined the effect of an anti-TREM-1 agonistic antibody (α-TREM-1) on colitis and identify its underlying mechanism of action. Administration of α-TREM-1 alleviated colitis in mice and resolved dysbiosis, which required TLR4/Myd88 signaling. α-TREM-1 increased the production of neutrophil extracellular traps and interleukin-22 by CD177+ neutrophils, which led to pathogen clearance and protection of the intestinal barrier. TREM-1 activation using an α-TREM-1 antibody protects against colitis by rebalancing the microbiota and protecting the epithelium against the immune response as well as modulates the function of neutrophils and macrophages. These results highlight the importance of the TREM-1 pathway in intestinal homeostasis and suggest that α-TREM-1 treatment may be an effective therapeutic strategy for inflammatory bowel disease.

Ciprofloxacin-encapsulated poly(DL-lactide-co-glycolide) nanoparticles and its antibacterial activity.

The aim of this study was to prepare ciprofloxacin HCl (CIP)-encapsulated poly(dl-lactide-co-glycolide) (PLGA) copolymer nanoparticles and its antibacterial potential was evaluated with pathogenic bacteria, Escherichia coli (E. coli), in vitro and in vivo. CIP-encapsulated nanoparticles of PLGA were prepared by multiple emulsion solvent evaporation method. PLGA nanoparticles showed spherical shapes with particle sizes around 100-300 nm. Loading efficiency was lower than 50% (w/w) because of water-solubility properties of CIP. At drug release study, CIP showed initial burst effect for 12 h and then continuously released for 2 weeks. At in vitro antibacterial activity test, CIP-encapsulated nanoparticles showed relatively lower antibacterial activity compared to free CIP due to the sustained release characteristics of nanoparticles. However, CIP-encapsulated PLGA nanoparticles (doses: 25 mg CIP/kg of mice) effectively inhibited the growth of bacteria due to the sustained release characteristics of nanoparticles, while free CIP was less effective on the inhibition of bacterial growth. These results indicated that CIP-encapsulated PLGA nanoparticles have superior effectiveness to inhibit the growth of bacteria in vivo.

Interleukin-33 regulates intestinal inflammation by modulating macrophages in inflammatory bowel disease.

Interleukin 33 (IL-33) that signals through the ST2 receptor has emerged as a critical modulator in several inflammatory disorders, including inflammatory bowel disease (IBD). However, the precise mechanisms by which IL-33 modulates IBD are controversial. The aim of this study was thus to clarify the role of IL-33 in IBD. The plasma levels of IL-33 were significantly decreased, but soluble ST2 levels were increased in patients with IBD compared to healthy individuals. Moreover, IL-33 restored goblet cell numbers and induced macrophage switching from the M1 to the M2 phenotype. These effects were sufficient to ameliorate colitis in dextran sodium sulfate, trinitrobenzene sulfonic acid, and peritoneal cavity cell transfer models. IL-33 facilitated goblet cell restoration via modulating macrophages toward the M2 phenotype. In addition, wound healing was significantly faster in IL-33-treated human monocyte-derived macrophages than in control cells, which could be attributed to increased polarisation into M2 macrophages. We found that patients with IBD show decreased serum levels of IL-33 compared with healthy individuals and that IL-33 can attenuate colitis and aid tissue repair in mice. The mechanism by which IL-33 exerts these effects appears to involve the stimulation of differentiation of goblet cells and M2 macrophages.

Development of a decellularization method to produce nerve allografts using less invasive detergents and hyper/hypotonic solutions.

BACKGROUND: Here, we describe a novel method of processing decellularized nerve grafts using osmotic effects of hypotonic and hypertonic solutions and Triton X-100 (a nonionic detergent) and CHAPS (an amphoteric detergent). MATERIALS AND METHODS: To evaluate decellularization, the devised method and Hudson's method were compared with respect to remaining cellular components (as assessed by H&E staining and S-100 immunoreactivity) and extracellular matrix structural integrity (as assessed by H&E staining and laminin immunoreactivity) by using rat sciatic nerves. In addition, a 1.5-cm sciatic nerve gap rat model was treated by implanting decellularized nerve grafts prepared using the devised method, Hudson's method, or an autograft to evaluate nerve regeneration. Nerve histomorphometry of distal stumps and wet muscle mass were evaluated at 12 weeks after implantation. RESULTS: The devised method produced outcomes similar to those of Hudson's method in terms of cellular component removal, but the devised method was significantly better in terms of ECM preservation. Histomorphometric study showed that the devised method produced significantly fewer nerve fiber and axonal densities than autografting, but much more than Hudson's method. The wet muscle mass of the devised method was also significantly lower than that of autografting, but much higher than that of Hudson's method. CONCLUSION: The described process for producing decellularized nerve grafts yielded better outcomes with respect to peripheral nerve regeneration than the established ionic detergent-based methods in a rat model. This study indicates that decellularized nerve grafts produced in this manner show favorable nerve regeneration used for bridging nerve gaps.

Interaction between postoperative shivering and hyperalgesia caused by high-dose remifentanil.

BACKGROUND: High-dose remifentanil-based anesthesia is associated with opioid-induced hyperalgesia (OIH) and postanesthetic shivering (PAS). These effects can be prevented by N-methyl-d-aspartate (NMDA) receptor antagonists. This study aimed to investigate correlations between OIH and PAS caused by high-dose remifentanil and the effects of low-dose ketamine on OIH and PAS. METHODS: Seventy-five patients scheduled for single-port laparoscopic gynecologic surgery were randomly allocated into three groups, each of which received intraoperative remifentanil: group L at 0.1 µg/kg/min; group H at 0.3 µg/kg/min; and group HK at 0.3 µg/kg/min plus 0.25 mg/kg ketamine just before incision, followed by a continuous infusion of 5 µg/kg/min ketamine until skin closure. RESULTS: PAS, postoperative tactile pain threshold, and the extent of hyperalgesia in group H were significantly different (P < 0.05) than in the other two groups. PAS was significantly correlated with OIH, including mechanically evoked pain such as postoperative tactile pain threshold (r = -0.529, P = 0.01) (r = -0.458, P = 0.021) and the extent of hyperalgesia (r = 0.537, P = 0.002) (r = 0.384, P = 0.031), respectively, in group H and group HK. Notably, both groups were treated with high-dose remifentanil. Tympanic membrane temperature, time to first postoperative analgesic requirement, postoperative pain scores, analgesic consumption, and cumulative patient-controlled analgesia volume containing morphine were comparable in all three groups. CONCLUSIONS: OIH, including the enhanced perception of pain, and PAS were both associated with high-dose remifentanil, were significantly correlated and were attenuated by a low dose of ketamine. This suggests that a common mechanism in part mediated through activation of the central glutamatergic system (e.g., NMDA receptors), underlies the two effects caused by high doses of remifentanil.

Successful treatment of abdominal cutaneous entrapment syndrome using ultrasound guided injection.

There are various origins for chronic abdominal pain. About 10-30% of patients with chronic abdominal pain have abdominal wall pain. Unfortunately, abdominal wall pain is not thought to be the first origin of chronic abdominal pain; therefore, patients usually undergo extensive examinations, including diagnostic laparoscopic surgery. Entrapment of abdominal cutaneous nerves at the muscular foramen of the rectus abdominis is a rare cause of abdominal wall pain. If abdominal wall pain is considered in earlier stage of chronic abdominal pain, unnecessary invasive procedures are not required and patients will reach symptom free condition as soon as the diagnosis is made. Here, we report a case of successful treatment of a patient with abdominal cutaneous nerve entrapment syndrome by ultrasound guided injection therapy.

A case of herpes zoster peripheral polyneuropathy manifested by foot drop in chronic myeloid leukemia.

In herpes zoster infection, neurological complications may be overlooked because pain is a more prominent symptom and because peripheral polyneuropathy associated with weakness is rare. A 57-year-old male visited our hospital, complaining of pain and skin eruptions on the right flank. He was diagnosed as having herpes zoster and the symptoms were alleviated by administration of acyclovir for a week. After three weeks, the herpes zoster relapsed. He was re-admitted and diagnosed with chronic myeloid leukemia (CML), and imatinib mesylate was prescribed for five weeks. Ten weeks after the onset of herpes zoster, bilateral foot drops and numbness of the right foot dorsum developed. Through an electrodiagnostic study, he was diagnosed as having peripheral polyneuropathy that was suspected to be caused by neural invasion by varicella zoster virus. After administration of famciclovir, not only the pain but also the neurologic symptoms improved. We herein report a case of peripheral polyneuropathy that was supposed to be related to herpes zoster.

A case of cephalic tetanus with unilateral ptosis and facial palsy.

Cephalic tetanus is defined as a combination of trismus and paralysis of one or more cranial nerves. Cranial nerves III, IV, VI, VII, and XII may be affected, but the facial nerve is most frequently implicated. A 64-year-old female visited hospital for left ptosis followed by facial palsy after a left forehead abrasion in a car accident. At nine days post injury, left ptosis developed, left facial palsy developed twelve days post injury, and at fifteen days post injury, trismus and dysphagia developed. The following day, there was progression of symptoms to generalized tetanus, such as dyspnea and generalized rigidity. Videofluoroscopic swallow study showed penetration and aspiration. We report a case of cephalic tetanus with ptosis, facial palsy, and dysphagia, which progressed to generalized tetanus.

The influence of laterality of pharyngeal bolus passage on Dysphagia in hemiplegic stroke patients.

OBJECTIVE: To investigate swallowing laterality in hemiplegic patients with stroke and recovery of dysphagia according to the laterality. METHOD: The sample was comprised of 46 dysphagic patients with hemiplegia after their first stroke. The sample's videofluoroscopic swallowing study (VFSS) was reviewed. Swallowing laterality was determined by the anterior-posterior view of VFSS. We measured width difference of barium sulfate liquid flow in the pharyngoesophageal segment. If there was double or more the width of that from the opposite width in the pharyngoesophageal segment more than twice on three trials of swallowing, then it was judged as having laterality. Subjects were assigned to no laterality (NL), laterality that is ipsilateral to hemiplegic side (LI), and laterality that is contralateral to hemiplegic side (LC) groups. We measured the following: prevalence of aspiration, the 8-point penetration-aspiration scale, and the functional dysphagia scale of the subjects at baseline and follow up. RESULTS: Laterality was observed in 45.7% of all patients. Among them, 52.4% were in the hemiplegic direction. There was no significant difference between groups at baseline in all measurements. When we compared the changes in all measurements on follow-up study, there were no significant differences between groups. CONCLUSION: Through this study, we found that there was no significant relation between swallowing laterality and the severity or prognosis of swallowing difficulty. More studies for swallowing laterality on stroke patients will be needed.

Skin temperature changes following sciatic nerve injury in rats.

In the clinical setting, skin temperature is both easily evaluated and useful in assessments of sympathetic dysfunction. The present study purposed to observe the serial skin temperature changes of both hindlimbs following several types of sciatic nerve injury (complete transection and ligation model [CTL], crush injury model [CRI], and chronic constriction injury model [CCI]) in Sprague-Dawley rats and, further, to delineate the possible mechanisms through various evaluation methods. The temperature differences between the intact and injured areas (ΔT) on the plantar surface and toes varied among the CTL, CRI, and CCI injury models during the acute stage (7 days post-injury). During the subacute to chronic stages (7-28 days post-injury), ΔT on the plantar area and toes of the CCI model were higher than those of the CTL and CRI models. The sciatic functional index was gradually restored in the CRI and CCI models, but was unchanged in the CTL model. The CTL model showed constant hypoesthesia; the CRI model, contrastingly, was restored to normal, and the CCI model showed gradual hyperesthesia until 28 days post-injury. The latency and amplitude of the compound muscle action potential (CMAP) in the involved plantar muscle was not found in the CTL group 4 weeks post-injury, but showed gradual restoration in the CRI and CCI models. Regression analysis revealed that the ΔT in the plantar area and toes were affected only by the CMAP amplitude in the involved plantar muscle; therefore, it can be said that the skin temperature on the injured area after sciatic nerve injury was influenced by the functional status of the involved muscle. Measurement of skin temperature can differentiate mild peripheral nerve injury from moderate-to-severe injuries, although its clinical significance might be limited.

Methotrexate-incorporated polymeric nanoparticles of methoxy poly(ethylene glycol)-grafted chitosan.

We prepared methotrexate (MTX)-encapsulated polymeric nanoparticles using methoxy poly(ethylene glycol) (MPEG)-grafted chitosan (ChitoPEG) copolymer. MTX-encapsulated polymeric nanoparticles of ChitoPEG copolymer has around 50-300nm in particle size and showed spherical shape when observed by transmission electron microscope (TEM). In (1)H nuclear magnetic resonance (NMR) study, the specific peaks of MTX and chitosan as a drug carrying inner-core disappeared at D(2)O and only the specific peak of MPEG was observed, while specific peaks of MPEG, MTX, and chitosan appeared in DCl/D(2)O mixtures. These results indicated that MTX was complexed with chitosan and then core-shell type nanoparticles had formed in aqueous environment, i.e., MTX/chitosan complexes composed of inner-core and MPEG composed of outer-shell of the nanoparticles. Loading efficiency of MTX in the polymeric nanoparticles was 94% (w/w) of ChitoPEG-1, 91.1% (w/w) of ChitoPEG-2, 90.1% (w/w) of ChitoPEG-3 and 65.2% (w/w) of ChitoPEG-4, expectively. The higher the drug feeding ratio, the higher the drug content and the lower the loading efficiency. The higher the MPEG graft ratio in the copolymer, the lower the drug content and loading efficiency. Drug contents evaluated by (1)H NMR were the same as found by UV spectrophotometer.