Early immune response of neuronal cells (U87) to heavy metal Cd or Pb exposure.

Heavy metals such as lead (Pb) and cadmium (Cd) exist as particulate matter (PM) in the air and can cause biological damage to cells, animals, and humans. However, the mechanism underlying the toxic effects of heavy metals on nerve cells has not yet been completely identified. Glioma is the most common and fatal tumor in the central nervous system; the U87 human glioblastoma cell line is commonly used when researching brain cancer, including aggressive malignant gliomas. Therefore, in this study, cell viability, cytotoxicity, and interleukin-6 (IL-6) levels were analyzed to confirm the effect of Cd and Pb exposure on U87 cells. On confirming the absence of significant effects on cell viability at low concentrations of heavy metals, Cd and Pb exposure had no effect on lactic acid dehydrogenase (LDH) activity at the concentrations (1 µg/L, 30 µg/L, and 1 mg/L) used in this study, and there was a remarkable effect of Cd and Pb exposure on the inflammatory response of these cells. Our findings provide a basis for future research elucidating the effects of heavy metal exposure on cellular pathology. Systematic studies with higher heavy metal concentrations and precision are warranted to deepen our understanding of the relationship between heavy metal exposure and neuronal responses.

An aptamer agonist of the insulin receptor acts as a positive or negative allosteric modulator, depending on its concentration.

Aptamers are widely used as binders that interact with targets with high affinity or as inhibitors of the function of target molecules. However, they have also been used to modulate target protein function, which they achieve by activating the target or stabilizing its conformation. Here, we report a unique aptamer modulator of the insulin receptor (IR), IR-A62. Alone, IR-A62 acts as a biased agonist that preferentially induces Y1150 monophosphorylation of IR. However, when administered alongside insulin, IR-A62 shows variable binding cooperativity depending on the ligand concentration. At low concentrations, IR-A62 acts as a positive allosteric modulator (PAM) agonist that enhances insulin binding, but at high concentrations, it acts as a negative allosteric modulator (NAM) agonist that competes with insulin for IR. Moreover, the concentration of insulin affects the binding of IR-A62 to IR. Finally, the subcutaneous administration of IR-A62 to diabetic mice reduces blood glucose levels with a longer-lasting effect than insulin administration. These findings imply that aptamers can elicit various responses from receptors beyond those of a simple agonist or inhibitor. We expect further studies of IR-A62 to help reveal the mechanism of IR activation and greatly expand the range of therapeutic applications of aptamers.

Laboratory investigations of herpes simplex virus-1 and -2 clinical samples in Korea.

OBJECTIVE: Herpes simplex virus (HSV) infections have been reported in 60% to 95% of the adult population worldwide, making them one of the most common infectious conditions globally. HSV-1 and HSV-2 cause severe disease in immunocompromised patients. Therefore, the aim of this study was to provide information that could be used to reduce the incidence of genital herpes caused by HSV-1 and HSV-2. METHODS: From September 2018 to December 2020, 59,381 specimens were collected from outpatients across primary and secondary hospitals in Korea who requested U2Bio (Korea) to conduct molecular biological testing of their samples for sexually transmitted infections. In this study, the positivity rates of HSV-1 and HSV-2 were analyzed according to age, sex, and specimen type. RESULTS: In the age-specific analysis of HSV-1, the highest positivity rate (0.58%) was observed in patients under 19 years of age, whereas the lowest positivity rate (0.08%) was observed in patients aged over 70 years. In the age-specific analysis of HSV-2, the highest positivity rate (2.53%) was likewise observed in patients under 19 years of age. CONCLUSION: Our study identified differences in the infection rates of HSV-1 and HSV-2 depending on patients' sex and age. These differences will be useful for improving disease prevention and control measures for HSV-1 and HSV-2.

Mycoplasma genitalium and Mycoplasma hominis infection in south Korea during 2018-2020.

BACKGROUND AND OBJECTIVES: Sexually transmitted infections (STIs) can remain undetected and untreated; therefore, rapid diagnosis and treatment of STIs are important. Mycoplasma genitalium (MG), Mycoplasma hominis (MH), and Ureaplasma urealyticum are sexually transmitted pathogens that cause asymptomatic, organ-specific, and chronic infections, thereby posing a threat to community health. Therefore, we investigated the epidemiological trends of MG and MH infections in South Korea for rapid diagnosis and treatment. MATERIALS AND METHODS: From September 2018 to December 2020, samples (catheter, pus, tissue, swab, and urine) were collected from outpatients of hospitals in South Korea for molecular biological venereal disease testing. DNA was extracted and analyzed using real-time polymerase chain reaction. RESULTS: Of the 59,381 samples analyzed, 8.78% (n=5,215) were positive for MG and MH. The MH positivity rate (5.51%, n=3,273) was higher than the MG positivity rate (3.27%, n=1,942). MG and MH positivity rates were the highest in patients aged <19 years. Men had higher MG positivity rate, whereas women had higher MH positivity rates. Furthermore, the MG-positivity rate was the highest in the swab samples of both men and women, whereas that of MH was the highest in the urine samples of men and swab samples of women. CONCLUSION: We identified the differences between MG and MH positivity rates based on sex, specimen, and age. Our findings can provide information for strategies that protect public health and reduce STI incidence and transmission.

Effects of climatic factors and particulate matter on Rotavirus A infections in Cheonan, Korea, in 2010-2019.

Rotavirus A is the most common cause of infectious diarrhea worldwide. This study aimed to retrospectively study and analyze 4009 stool samples that were tested for viruses causing diarrhea, using multiplex reverse transcription PCR at Dankook University Hospital between 2010 and 2019. Furthermore, we determined the correlation between these factors and various climatic factors, including wind-chill temperature, relative humidity, rate of sunshine, and particulate matter. Rotavirus A infections occurred frequently in February, March, and April on an annual basis. Furthermore, during the study, the detection rate was highest at 17.0% (n=61/359) in 2011. Based on an analysis of weather big data, patient age, and period-specific infection during the summer, when the wind-chill temperature and relative humidity were high, the Rotavirus A infection rate was very low. Relative humidity (p=0.020) and particulate matter (p=0.049) were associated with the average number of monthly cases of Rotavirus A infection. However, wind chill temperature (p=0.074) and rate of sunshine (p=0.993) were not associated with the average monthly distribution of Rotavirus A cases. These results indicate that Rotavirus A infection was correlated with relative humidity and particulate matter during the study period and further the current understanding of the distribution of Rotavirus A infections resulting from climatic factors and particulate matter. This could help establish climate-related health policies to reduce the incidence of diarrhea and guide the development of vaccines against Rotavirus A.

Interrelationship between climatic factors and incidence of FBD caused by Clostridioides difficile toxin B, Clostridium perfringens, Campylobacter spp., and Escherichia coli O157:H7.

Foodborne diseases (FBDs) remain a global public health concern. Climatic factors such as wind-chill temperature, rainfall, and relative humidity affect the incidence of several FBDs. This study was performed to analyze how the various factors of the climate influence the incidence and severity of FBDs. This study retrospectively analyzed the results of multiplex polymerase chain reaction (mPCR) tests for diarrhea-causing bacteria performed on 2300 fecal samples obtained from patients at Dankook University Hospital, Cheonan, from June 2010 to December 2019. The Clostridioides difficile toxin B infection rate positively correlated with the intensity of sunshine, and the content of particulate matter. The Campylobacter spp. infection rate positively correlated with wind-chill temperature and the content of particulate matter. The Escherichia coli O157:H7 infection rate positively correlated with relative humidity. These findings may explain the dynamics and risks of Clostridioides difficile toxin B, Clostridium perfringens, Campylobacter spp., and Escherichia coli O157:H7 infection. They may help predict interrelationships among climatic factors and standardize national environmental health policies. However, in-depth research with large-scale data, molecular biology, and epidemiology would be required going forward. Future research would also require objective indicators of the changes in the prevalence of FBD-causing microbial pathogens for the effective prevention and management of these bacterial infections.

Effects of climatic factors on human parainfluenza 1, 2, and 3 infections in Cheonan, Republic of Korea.

Studying relationships between meteorological conditions and respiratory virus infections may help interpret the causality of disease outbreaks and provide a better understanding of the seasonal distribution of viruses. Therefore, in this study, we analyzed the correlations between meteorological data and the trends of infection by human parainfluenza virus-1 (HPIV-1; also known as human respirovirus 1), human parainfluenza virus-2 (human orthorubulavirus 2), and human parainfluenza virus-3 (human respirovirus 3) using 9010 viral samples collected at Dankook University Hospital from January 1, 2012, to December 31, 2018. Infection frequency data were used to detect the seasonal patterns of HPIV-1, HPIV-2, and HPIV-3 infections, and these patterns were compared with local weather data over the same period. We performed descriptive statistical analysis, frequency analysis, t test, and binomial logistic regression analysis to examine the relationships of weather and particulate matter conditions with the incidence of HPIV-1, HPIV-2, and HPIV-3 infections. The highest average infection rate with one of these three viruses (88.17%) was found in children aged 1-9 years. Specifically, the infection rate of HPIV-1 was 91.9% in children aged 1-9 years, whereas that of HPIV-2 and HPIV-3 was 86.3%. HPIV infection exhibited a meaningful relationship with climatic factors, such as temperature, wind-chill temperature, and atmospheric pressure. Our results suggest that climate changes might affect the rate of infection by HPIV. These findings may help in predicting the effectiveness of preventive strategies of HPIV infection.

Effect of task-oriented activities on hand functions, cognitive functions and self-expression of elderly patients with dementia.

[Purpose] This study investigates the effects of task-oriented activities on hand function, cognitive function, and self-expression of the elderly with dementia, and then identify the influencing factors on self-expression in sub-factors of dependent variables. [Subjects and Methods] Forty elderly persons were divided into two groups: intervention group (n=20) and control group (n=20). The interventions were applied to the subjects 3 times a week, 50 minutes per each time, for a total of five weeks. We measured the jamar hand dynamometer test for grip strength, the jamar hydraulic pinch gauge test for prehension test, nine-hole pegboard test for coordination test, and Loewenstein Occupational Therapy Cognitive Assessment-Geriatric Population for cognitive function, and self-expression rating scale for self-expression test. [Results] The task-oriented activities promoted hand function, cognitive function (visual perception, spatial perception, visuomotor organization, attention & concentration) and self-expression of the elderly with early dementia, and the factors influencing the self-expression were cognitive function (visual perception) and hand function (coordination). The study showed that the task-oriented program enabled self-expression by improving hand function and cognitive function. [Conclusion] This study suggested that there should be provided the task-oriented program for prevention and treatment of the elderly with early dementia in the clinical settings and it was considered that results have a value as basic data that can be verified relationship of hand function, cognitive function, and self-expression.

Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14.

Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS assay, and cell death signaling was explored by immunoblot analysis. Fluorescence-activated cell sorting was performed to evaluate the affinity and specificity of AS1411-aptamers in SNU-761 HCC cells. We investigated the in vivo effects of the AS1411-aptamer using BALB/c nude mice in a subcutaneous xenograft model with SNU-761 cells. Treatment with a modified AS1411-aptamer significantly decreased in vitro (under normoxic [P = 0.035] and hypoxic [P = 0.018] conditions) and in vivo (under normoxic conditions, P = 0.041) HCC cell proliferation compared to control aptamers. AS1411- and control aptamers failed to control HCC cell proliferation. However, AS1411- and the modified AS1411-aptamer did not induce caspase activation. Decrease in cell growth by AS1411 or modified AS1411 was not prevented by caspase or necrosis inhibitors. In a microarray, AS1411 significantly enhanced galectin-14 expression. Suppression of HCC cell proliferation by the modified AS1411-aptamer was attenuated by galectin-14 siRNA transfection. Modified AS1411-aptamer suppressed HCC cell growth in vitro and in vivo by up-regulating galectin-14 expressions. Modified AS1411-aptamers may have therapeutic potential as a novel targeted therapy for HCC.

A Novel Role of Serotonin Receptor 2B Agonist as an Anti-Melanogenesis Agent.

BW723C86, a serotonin receptor 2B agonist, has been investigated as a potential therapeutic for various conditions such as anxiety, hyperphagia and hypertension. However, the functional role of BW723C86 against melanogenesis remains unclear. In this study, we investigate the effect of serotonin receptor 2B (5-HTR2B) agonist on melanogenesis and elucidate the mechanism involved. BW723C86 reduced melanin synthesis and intracellular tyrosinase activity in melan-A cells and normal human melanocytes. The expression of melanogenesis-related proteins (tyrosinase, TRP-1 and TRP-2) and microphthalmia-associated transcription factor (MITF) in melan-A cells decreased after BW723C86 treatment. The promoter activity of MITF was also reduced by BW723C86 treatment. The reduced level of MITF was associated with inhibition of protein kinase A (PKA) and cAMP response element-binding protein (CREB) activation by BW723C86 treatment. These results suggest that the serotonin agonist BW723C86 could be a potential therapeutic agent for skin hyperpigmentation disorders.

Enhanced compatibility and initial stability of Ti6Al4V alloy orthodontic miniscrews subjected to anodization, cyclic precalcification, and heat treatment.

OBJECTIVE: To evaluate the bioactivity, and the biomechanical and bone-regenerative properties of Ti6Al4V miniscrews subjected to anodization, cyclic precalcification, and heat treatment (APH treatment) and their potential clinical use. METHODS: The surfaces of Ti6Al4V alloys were modified by APH treatment. Bioactivity was assessed after immersion in simulated body fluid for 3 days. The hydrophilicity and the roughness of APH-treated surfaces were compared with those of untreated (UT) and anodized and heat-treated (AH) samples. For in vivo tests, 32 miniscrews (16 UT and 16 APH) were inserted into 16 Wistar rats, one UT and one APH-treated miniscrew in either tibia. The miniscrews were extracted after 3 and 6 weeks and their osseointegration (n = 8 for each time point and group) was investigated by surface and histological analyses and removal torque measurements. RESULTS: APH treatment formed a dense surface array of nanotubular TiO2 layer covered with a compact apatite-like film. APH-treated samples showed better bioactivity and biocompatibility compared with UT and AH samples. In vivo, APH-treated miniscrews showed higher removal torque and bone-to-implant contact than did UT miniscrews, after both 3 and 6 weeks (p < 0.05). Also, early deposition of densely mineralized bone around APH-treated miniscrews was observed, implying good bonding to the treated surface. CONCLUSIONS: APH treatment enhanced the bioactivity, and the biomechanical and bone regenerative properties of the Ti6Al4V alloy miniscrews. The enhanced initial stability afforded should be valuable in orthodontic applications.

Theranostic systems assembled in situ on demand by host-guest chemistry.

Theranostic systems have been explored extensively for a diagnostic therapy in the forms of polymer conjugates, implantable devices, and inorganic nanoparticles. In this work, we report theranostic systems in situ assembled by host-guest chemistry responding to a request. As a model theranostic system on demand, cucurbit[6]uril-conjugated hyaluronate (CB[6]-HA) was synthesized and decorated with FITC-spermidine (spmd) and/or formyl peptide receptor like 1 (FPRL1) specific peptide-spmd by simple mixing in aqueous solution. The resulting (FITC-spmd and/or peptide-spmd)@CB[6]-HA was successfully applied to the bioimaging of its target-specific delivery to B16F1 cells with HA receptors and its therapeutic signal transduction with elevated Ca(2+) and phosphor-extracellular signal-regulated kinase (pERK) levels in FPRL1-expressing human breast adenocarcinoma (FPRL1/MCF-7) cells. Finally, we could confirm in vitro and in vivo stability of the highly specific host-guest interaction. The on-demand theranostic platform technology using host-guest chemistry can be exploited for various bioimaging, biosensing, drug delivery, and tissue engineering applications.

Anti-Flt1 peptide - hyaluronate conjugate for the treatment of retinal neovascularization and diabetic retinopathy.

Anti-angiogenic therapeutics has been investigated extensively for the treatment of retinal and choroidal vascular diseases, and diabetic retinopathy. Anti-Flt1 peptide of GNQWFI is an antagonistic peptide for vascular endothelial growth factor receptor 1 (VEGFR1 or Flt1) inhibiting VEGFR1-mediated endothelial cell migration and tube formation. In this work, anti-Flt1 peptide (GGNQWFI) was chemically conjugated to tetra-n-butyl ammonium modified hyaluronate (HA-TBA) via amide bond formation in dimethyl sulfoxide (DMSO) using benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). The resulting HA - GGNQWFI conjugate self-assembled to form micelle-like nanoparticles in aqueous solution, as confirmed and characterized by transmission electron microscopy (TEM). According to in vitro biological activity tests, HA - GGNQWFI conjugate exhibited a dose-dependent inhibition effect on the binding of Flt1-Fc to VEGF(165) coated on the well. Furthermore, anti-Flt1 peptide - HA conjugate effectively inhibited retinal choroidal neovascularization (CNV) in laser induced CNV model rats. The retinal vascular permeability and the deformation of retinal vascular structure were also significantly reduced in diabetic retinopathy model rats after treatment with anti-Flt1 peptide - HA conjugate. Pharmacokinetic analysis confirmed the increased mean residence time of anti-Flt1 peptide after conjugation to HA longer than 2 weeks.

Single-file diffusion of protein drugs through cylindrical nanochannels.

A new drug delivery device using cylindrical block copolymer nanochannels was successfully developed for controlled protein drug delivery applications. Depending on the hydrodynamic diameter of the protein drugs, the pore size in cylindrical nanochannels could be controlled precisely down to 6 nm by Au deposition. Zero-order release of bovine serum albumin (BSA) and human growth hormone (hGH) by single-file diffusion, which has been observed for gas diffusion through zeolite pores, was realized up to 2 months without protein denaturation. Furthermore, a nearly constant in vivo release of hGH from the drug delivery nanodevice implanted to Sprague-Dawley (SD) rats was continued up to 3 weeks, demonstrating the feasibility for long-term controlled delivery of therapeutic protein drugs.

Long acting hyaluronate--exendin 4 conjugate for the treatment of type 2 diabetes.

Despite clinical exploitation of exendin 4 for the treatment of type 2 diabetes, the significantly short half-life requiring twice a day injection has limited the wide applications. In this work, a protocol for the synthesis of long acting hyaluronate (HA) - exendin 4 conjugate was successfully developed using Michael addition chemistry between vinyl sulfone modified HA (HA-VS) and thiolated exendin 4. The exendin 4 content could be controlled in the range of 5-30 molecules per single HA chain with a bioconjugation efficiency higher than 90%. The conjugation of exendin 4 with HA resulted in about 20 times improved in vitro serum stability maintaining the hypoglycemic and gluco-regulatory bioactivities of exendin 4. HA - exendin 4 conjugates showed excellent glucose-lowering capabilities in type 2 db/db mice demonstrating protracted hypoglycemic effect up to 3 days after a single subcutaneous injection. Furthermore, insulin immunohistochemical analysis of islets in db/db mice confirmed the improved insulinotropic activity of HA - exendin 4 conjugates. The HA - exendin 4 conjugates will be investigated further as a twice a week injection dosage form for clinical applications.

Target specific and long-acting delivery of protein, peptide, and nucleotide therapeutics using hyaluronic acid derivatives.

Hyaluronic acid (HA) is a biodegradable, biocompatible, non-toxic, non-immunogenic and non-inflammatory linear polysaccharide, which has been used for various medical applications such as arthritis treatment, ocular surgery, tissue augmentation, and so on. In this review, the effect of chemical modification of HA on its distribution throughout the body was reported for target specific and long-acting delivery applications of protein, peptide, and nucleotide therapeutics. According to the real-time bio-imaging of HA derivatives using quantum dots (QDot), HA-QDot conjugates with 35mol% HA modification maintaining enough binding sites for HA receptors were mainly accumulated in the liver, while those with 68mol% HA modification losing much of HA characteristics were evenly distributed to the tissues in the body. The results are well matched with the fact that HA receptors are abundantly present in the liver with a high specificity to HA molecules. Accordingly, slightly modified HA derivatives were used for target specific intracellular delivery of nucleotide therapeutics and highly modified HA derivatives were used for long-acting conjugation of peptide and protein therapeutics. HA has been also used as a novel depot system in the forms of physically and chemically crosslinked hydrogels for various protein drug delivery. This review will give you a peer overview on novel HA derivatives and the latest advances in HA-based drug delivery systems of various biopharmaceuticals for further clinical development.

Synthesis, characterization, and preliminary assessment of anti-Flt1 peptide-hyaluronate conjugate for the treatment of corneal neovascularization.

Anti-Flt1 peptide of GNQWFI has been reported to inhibit vascular endothelial growth factor receptor 1 (VEGFR1) - mediated endothelial cell migration and tube formation. In this work, a protocol to synthesize anti-Flt1 peptide-hyaluronate (HA) conjugate was successfully developed for the treatment of corneal neovascularization. Using tetrabutyl ammonium salt of HA (HA-TBA), water-insoluble anti-Flt1 peptide could be conjugated with HA in dimethyl sulfoxide (DMSO) by the amide bond formation between carboxyl groups of HA and N-terminal amine groups of GGNQWFI. The formation of anti-Flt1 peptide-HA conjugate was confirmed by (1)H NMR and fluorometric analyses. The average number of grafted peptide molecules in anti-Flt1 peptide-HA conjugates could be controlled from 3 to 30 per single HA chain by changing the feeding amount of peptide for the conjugation reaction. According to in vitro biological activity tests, anti-Flt1 peptide-HA conjugate exhibited a significant inhibition effect on the binding of Flt1-Fc to VEGF(165) coated on the well. Furthermore, in vivo biological activity of anti-Flt1 peptide-HA conjugate was confirmed from the inhibitory effect on corneal neovascularization in silver nitrate cauterized corneas of SD rats. The VEGF receptor 2 expression was also reduced after treatment with anti-Flt1 peptide-HA conjugate. The water-soluble anti-Flt1 peptide-HA conjugate was thought to have a potential to be developed as anti-angiogenic therapeutics for the treatment of corneal neovascularization.

Guided bone regeneration by poly(lactic-co-glycolic acid) grafted hyaluronic acid bi-layer films for periodontal barrier applications.

A novel protocol for the synthesis of biocompatible and degradation controlled poly(lactic-co-glycolic acid) grafted hyaluronic acid (HA-PLGA) was successfully developed for periodontal barrier applications. HA was chemically modified with adipic acid dihydrazide (ADH) in the mixed solvent of water and ethanol, which resulted in a high degree of HA modification up to 85 mol.%. The stability of HA-ADH to enzymatic degradation by hyaluronidase increased with ADH content in HA-ADH. When the ADH content in HA-ADH was higher than 80 mol.%, HA-ADH became soluble in dimethyl sulfoxide and could be grafted to the activated PLGA with N,N'-dicyclohexyl carbodiimide and N-hydroxysuccinimide. The resulting HA-PLGA was used for the preparation of biphasic periodontal barrier membranes in chloroform. According to in vitro hydrolytic degradation tests in phosphate buffered saline, HA-PLGA/PLGA blend film with a weight ratio of 1/2 degraded relatively slowly compared to PLGA film and HA coated PLGA film. Four different samples of a control, OSSIX(TM) membrane, PLGA film, and HA-PLGA/PLGA film were assessed as periodontal barrier membranes for the calvarial critical size bone defects in SD rats. Histological and histomorphometric analyses revealed that HA-PLGA/PLGA film resulted in the most effective bone regeneration compared to other samples with a regenerated bone area of 63.1% covering the bone defect area.

Signal transduction of hyaluronic acid-peptide conjugate for formyl peptide receptor like 1 receptor.

Agonistic and antagonistic peptides for formyl peptide receptor like 1 (FPRL1) receptor have been investigated as novel drug candidates for inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis. In this work, a novel protocol for the synthesis of hyaluronic acid (HA)-peptide (CWRYMVm) conjugate for FPRL1 receptor was successfully developed for further clinical applications of peptide drugs. Aminoethyl methacrylated HA (HAAEMA) was synthesized by the coupling reaction of tetrabutyl ammonium salt of HA (HA-TBA) and AEMA using benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP) in dimethyl sulfoxide (DMSO). Then, HA-AEMA was conjugated with CWRYMVm in water via Michael addition reaction between methacrylate group of HA-AEMA and thiol group in cysteine. The formation of HA-peptide conjugate was confirmed by 1H NMR and gel permeation chromatography (GPC). The average number of conjugated peptide molecules could be controlled from 5 to 23 per single HA chain. The HA-peptide conjugate showed serum stability longer than four days. In Vitro signal transduction activity of the HA-peptide conjugate for FPRL1 receptor was confirmed from the elevated levels of phospho-extracellular signal-regulated kinase (pERK) and calcium ion in FPRL1 overexpressing RBL-2H3 cells. The partially decreased biological activity of HA-peptide conjugates by the steric hindrance of HA was recovered after its degradation by hyaluronidase treatment.

Hyaluronic acid-polyethyleneimine conjugate for target specific intracellular delivery of siRNA.

A novel target specific small interfering RNA (siRNA) delivery system was successfully developed using polyethyleneimine (PEI)-hyaluronic acid (HA) conjugate. Anti-PGL3-Luc siRNA was used as a model system suppressing the PGL3-Luc gene expression. The siRNA/PEI-HA complex with an average size of ca. 21 nm appeared to be formed by electrostatic interaction between the negatively charged siRNA and the positively charged PEI of PEI-HA conjugate. The cytotoxicity of siRNA/PEI-HA complex to B16F1 cells was lower than that of siRNA/PEI complex according to the MTT assay. When B16F1 and HEK-293 cells were treated with fluorescein isothiocyanate (FITC) labeled siRNA/PEI-HA complex, B16F1 cells, with a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), showed higher green fluorescent intensity than HEK-293 cells because of the HA receptor mediated endocytosis of the complex. Accordingly, the PGL3-Luc gene silencing of anti-PGL3-Luc siRNA/PEI-HA complex was more efficient in B16F1 cells than in HEK-293 cells. In addition, the inhibited PGL3-Luc gene silencing effect in the presence of free HA in the transfection medium revealed that siRNA/HA-PEI complex was selectively taken up to B16F1 cells via HA receptor mediated endocytosis. All these results demonstrated that the intracellular delivery of anti-PGL3-Luc siRNA/PEI-HA complex could be facilitated by the HA receptor mediated endocytosis.