Clinical Characteristics of Cryopyrin-Associated Periodic Syndrome and Long-Term Real-World Efficacy and Tolerability of Canakinumab in Japan: Results of a Nationwide Survey.

OBJECTIVE: We assess the clinical characteristics of patients with cryopyrin-associated periodic syndrome (CAPS) in Japan and evaluate the real-world efficacy and safety of interleukin-1 (IL-1) inhibitors, primarily canakinumab. METHODS: Clinical information was collected retrospectively, and serum concentrations of canakinumab and cytokines were analyzed. RESULTS: A total of 101 patients were included, with 86 and 15 carrying heterozygous germline and somatic mosaic mutations, respectively. We identified 39 mutation types, and the common CAPS-associated symptoms corresponded with those in previous reports. Six patients (5.9% of all patients) died, with four of the deaths caused by CAPS-associated symptoms. Notably, 73.7% of patients (100%, 79.6%, and 44.4% of familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and chronic infantile neurological cutaneous articular syndrome/neonatal onset multisystem inflammatory disease, respectively) achieved complete remission with canakinumab, and early therapeutic intervention was associated with better auditory outcomes. In some patients, canakinumab treatment stabilized the progression of epiphysial overgrowth and improved height gain, visual acuity, and renal function. However, 23.7% of patients did not achieve inflammatory remission with crucial deterioration of organ damage, with two dying while receiving high-dose canakinumab treatment. Serological analysis of canakinumab and cytokine concentrations revealed that the poor response was not related to canakinumab shortage. Four inflammatory nonremitters developed inflammatory bowel disease (IBD)-unclassified during canakinumab treatment. Dual biologic therapy with canakinumab and anti-tumor necrosis factor-α agents was effective for IBD- and CAPS-associated symptoms not resolved by canakinumab monotherapy. CONCLUSION: This study provides one of the largest epidemiologic data sets for CAPS. Although early initiation of anti-IL-1 treatment with canakinumab is beneficial for improving disease prognosis, some patients do not achieve remission despite a high serum concentration of canakinumab. Moreover, IBD may develop in CAPS after canakinumab treatment.

Rheumatoid factor value for determining the first biologic agent to use for non-systemic juvenile idiopathic arthritis.

OBJECTIVES: Currently, no indicators on which biologic disease-modifying anti-rheumatic drugs (bDMARDs) should be used first for juvenile idiopathic arthritis (JIA) have been established. Thus, this study aimed to determine the useful biomarkers in JIA to enable the best selection of the first bDMARDs without primary failure. METHODS: This retrospective study used data of patients examined for JIA between 2015 and 2021 at Kagoshima University Hospital in Japan. RESULTS: Altogether, 67 cases of non-systemic JIA were analyzed, excluding cases that had been treated for <6 months. Of the 67 cases, 52 were treated with bDMARDs and all rheumatoid factor (RF)+ types (32 cases) were treated with bDMARDs. Eleven cases (31.4&) (all were RF+ types and used anti-tumor necrosis factor (TNF)α agents) switched to other bDMARDs because of primary failure, and nine cases had secondary failure (6;anti-TNF, 3;anti-Interleukin-6). A significant difference in pre-treatment RF values (177.9 vs 25.7 IU/ml, p = 0.002) and presence (Odds Ratio 1.952,p = 0.004) were observed between the primary failure group and effective group. CONCLUSIONS: RF+ JIA required bDMARDs with high probability. JIA with high titre of RF tends to be refractory to anti-TNFα agents. Tocilizumab or abatacept could be a first-choice bDMARD in such cases.

Re-examination of Peptide-Sequence-Dependent Gene Expression of Cysteine-Installed Pegylated Oligolysine/DNA Complexes.

We previously synthesized cysteine-installed C-terminally PEGylated oligolysines with 20 amino acid residues to form cross-linked polymeric micelles (PMs) with luciferase-coding plasmid DNA as a candidate for artificial gene vectors. Luciferase gene expression in HeLa cells mediated by PEG-CK18C, PEG-CK9CK9, and PEG-K9CK9C was reported to be 35-, 5.4-, and 1.3-fold higher than that mediated by cysteine-uninstalled PEGylated oligolysine PEG-K20, respectively. However, after the publication, the survival rate of HeLa cells used in the previous study was found to be lower than usual when subcutaneously implanted into mice to create a xenograft model. In this study, to re-examine the peptide sequence-dependent gene expression, gene expression efficacy mediated by PEG-peptide PMs was compared with the PM cellular uptake results using newly obtained HeLa cell lines and the additional cell lines Huh-7, PANC-1, and BxPC3. As a result, PEG-K9CK9C PMs mediated the maximum gene expression in all cell lines, and the corresponding cellular uptake was also obtained. Therefore, we concluded that our previous results were erroneously obtained due to normality-depleted HeLa cells. A comparison of physicochemical characterizations, gene expression efficacy, and cellular uptake of PEG-peptide PMs is discussed in detail.

Fine-tuning of polyaspartamide derivatives with alicyclic moieties for systemic mRNA delivery.

Development of efficient delivery vehicles for in vitro transcribed mRNA (IVT mRNA) is currently a major challenge in nanomedicines. For systemic mRNA delivery, we developed a series of cationic amphiphilic polyaspartamide derivatives (PAsp(DET/R)s) carrying various alicyclic (R) moieties with diethylenetriamine (DET) in the side chains to form mRNA-loaded polyplexes bearing stability under physiological conditions and possessing endosomal escape functionality. While the size and ζ-potential of polyplexes were comparable among various PAsp(DET/R)s, the transfection efficiencies of polyplexes were considerably varied due to difference in the R moieties of PAsp(DET/R)s and were described by an octanol-water (or buffer at pH 7.3) distribution coefficient (logD7.3). The critical logD7.3 for the efficient in vitro transfection of mRNA was indicated at -2.7 to -1.8. The polyplexes with logD7.3 > -1.8 elicited the much higher in vitro transfection efficiencies. After systemic administration, the polyplexes with logD7.3 from -1.8 to -1.3 elicited the significant mRNA expression specifically in the lungs. The highest mRNA expression in the lungs was achieved by a polyaspartamide derivative having a cyclohexylethyl group (PAsp(DET/CHE)), which induced more than 10-fold increase in mRNA transfection efficiency compared to commercially available lipid nanoparticles. The higher mRNA expression by polyplexes in the lungs was explained well by the preferential lung accumulation of intact mRNA, as determined by quantitative real-time PCR. Our results demonstrate that PAsp(DET/R)s are a promising synthetic material for the enhanced systemic IVT mRNA delivery.

Anti-TNF treatment corrects IFN-γ-dependent proinflammatory signatures in Blau syndrome patient-derived macrophages.

BACKGROUND: Blau syndrome (BS) is an autoinflammatory disease associated with mutations in nucleotide-binding oligomerization domain 2. Although treatments with anti-TNF agents have been reported to be effective, the underlying molecular mechanisms remain unclear. OBJECTIVE: We aimed to elucidate the mechanisms of autoinflammation in patients with BS and to clarify how anti-TNF treatment controls the disease phenotype at the cellular level in clinical samples. METHODS: Macrophages were differentiated from monocytes of 7 BS patients, and global transcriptional profiles of 5 patients were analyzed with or without IFN-γ stimulation. Macrophages were also generated from BS-specific induced pluripotent stem cells (iPSCs), and their transcriptome was examined for comparison. RESULTS: Aberrant inflammatory responses were observed upon IFN-γ stimulation in macrophages from untreated BS patients, but not in those from patients treated with anti-TNF. iPSC-derived macrophages carrying a disease-associated mutation also showed IFN-γ-dependent accelerated inflammatory responses. Comparisons of peripheral blood- and iPSC-derived macrophages revealed the upregulation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) targets in unstimulated macrophages as a common feature. CONCLUSIONS: IFN-γ stimulation is one of the key signals driving aberrant inflammatory responses in BS-associated macrophages. However, long-term treatment with anti-TNF agents ameliorates such abnormalities even in the presence of IFN-γ stimulation. Our data thus suggest that preexposure to TNF or functionally similar cytokines inducing NF-κB-driven proinflammatory signaling during macrophage development is a prerequisite for accelerated inflammatory responses upon IFN-γ stimulation in BS.

Dynamic Stabilization of Unit Polyion Complexes Incorporating Small Interfering RNA by Fine-Tuning of Cationic Block Length in Two-Branched Poly(ethylene glycol)-b-poly(l-lysine).

To stabilize small interfering RNA (siRNA) in the bloodstream for systemic RNAi therapeutics, we previously fabricated ultrasmall siRNA nanocarriers that were sub-20 nm in hydrodynamic diameter, named as unit polyion complexes (uPICs), using two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The blood retention time of uPICs is dramatically increased in the presence of free bPEG-PLys, suggesting dynamic stabilization of uPICs by free bPEG-PLys based on their equilibrium. Herein, we examined how the degree of polymerization of PLys (DPPLys) affected the dynamic stability of uPICs in the bloodstream during prolonged circulation. We prepared a series of bPEG-PLys with DPPLys values of 10, 13, 20, 40, and 80 for the uPIC formation and siRNA with 40 negative charges. These bPEG-PLys were then evaluated in physicochemical characterization and pharmacokinetic analyses. Structural analyses revealed that the uPIC size and association numbers were mainly determined by the molecular weights of PEG and DPPLys, respectively. Under bPEG-PLys-rich conditions, the hydrodynamic diameters of uPICs were 15-20 nm, which were comparable to that of the bPEG block (i.e., ∼18 nm). Importantly, DPPLys significantly affected the association constant of bPEG-PLys to siRNA (Ka) and blood retention of free bPEG-PLys. A smaller DPPLys resulted in a lower Ka and a longer blood retention time of free bPEG-PLys. Thus, DPPLys can control the dynamic stability of uPICs, i.e., the balance between Ka and blood concentration of free bPEG-PLys. Ultimately, the bPEG-PLys with DPPLys values of 14 and 19 prolonged the blood circulation of siRNA-loaded uPICs with relatively small amounts of free bPEG-PLys. This study revealed that the uPIC formation between siRNA and bPEG-PLys can be controlled by their charges, which may be helpful for designing PIC-based delivery systems.

Clinical impact of myositis-specific autoantibodies on long-term prognosis of juvenile idiopathic inflammatory myopathies: multicentre study.

OBJECTIVES: This study aimed to investigate the clinical characteristics, treatment and prognosis of juvenile idiopathic inflammatory myopathies (JIIM) in Japan for each myositis-specific autoantibody (MSA) profile. METHODS: A multicentre, retrospective study was conducted using data of patients with JIIM at nine paediatric rheumatology centres in Japan. Patients with MSA profiles, determined by immunoprecipitation using stored serum from the active stage, were included. RESULTS: MSA were detected in 85 of 96 cases eligible for the analyses. Over 90% of the patients in this study had one of the following three MSA types: anti-melanoma differentiation-associated protein 5 (MDA5) (n = 31), anti-transcriptional intermediary factor 1 alpha and/or gamma subunits (TIF1γ) (n = 25) and anti-nuclear matrix protein 2 (NXP2) (n = 25) antibodies. Gottron papules and periungual capillary abnormalities were the most common signs of every MSA group in the initial phase. The presence of interstitial lung disease (ILD) was the highest risk factor for patients with anti-MDA5 antibodies. Most patients were administered multiple drug therapies: glucocorticoids and MTX were administered to patients with anti-TIF1γ or anti-NXP2 antibodies. Half of the patients with anti-MDA5 antibodies received more than three medications including i.v. CYC, especially patients with ILD. Patients with anti-MDA5 antibodies were more likely to achieve drug-free remission (29 vs 21%) and less likely to relapse (26 vs 44%) than others. CONCLUSION: Anti-MDA5 antibodies are the most common MSA type in Japan, and patients with this antibody are characterized by ILD at onset, multiple medications including i.v. CYC, drug-free remission, and a lower frequency of relapse. New therapeutic strategies are required for other MSA types.

External validation of the EULAR/ACR idiopathic inflammatory myopathies classification criteria with a Japanese paediatric cohort.

OBJECTIVES: To assess the performance of the EULAR/ACR idiopathic inflammatory myopathies (IIMs) classification criteria to classify juvenile IIMs (JIIMs) in an Asian paediatric population. METHODS: Sixty-eight JIIM patients and 49 non-JIIM patients diagnosed at seven major paediatric rheumatology centres in Japan between 2008 and 2015 were enrolled. Retrospective data were collected, and each patient's data form was submitted. The expert group reviewed the forms and re-examined the diagnoses. The EULAR/ACR criteria were then applied and the probability of having JIIM was determined for each case. The sensitivity and specificity of the EULAR/ACR criteria were compared with those of other existing criteria. RESULTS: The sensitivity/specificity of the EULAR/ACR classification criteria were 92.1/100% with muscle biopsy data (n = 38); 86.7/100% without muscle biopsy data (n = 30) and 89.7/100% in our total cohort (n = 68). The sensitivity of Bohan and Peter's criteria and Tanimoto's criteria were 80.9 and 64.7% in our total cohort, respectively. Among 68 physician-diagnosed JIIM patients, seven cases (three JDM and four overlap myositis) were not classified as JIIM because the probability did not reach the cut-off point (55%). The three JDM patients all presented with only one of the three skin manifestations that are listed in the criteria: Gottron's sign. CONCLUSION: Our validation study with Japanese JIIM cases indicates that the EULAR/ACR classification criteria for IIM generally perform better than existing diagnostic criteria for myositis.

A case of cryopyrin-associated periodic fever syndrome during canakinumab administration complicated by inflammatory bowel disease.

Cryopyrin-associated periodic fever syndrome (CAPS) is a highly debilitating disorder, which is characterized by unregulated interleukin-1ß production driven by autosomal dominantly inherited mutations in the NLRP3 gene. Patients with CAPS often present with early-onset episodes of fever and rash. These patients also present with variable systemic signs and symptoms, such as arthritis, sensorineural hearing loss, chronic aseptic meningitis, and skeletal abnormalities, but minimal gastrointestinal symptoms. Recently, effective therapies for CAPS targeted against interleukin-1 have become available. We report a case of a young Japanese woman with CAPS who developed inflammatory bowel disease during canakinumab therapy. The patient had colostomy after intestinal perforation and changed canakinumab to infliximab. To the best of our knowledge, this is the first report of a case of inflammatory bowel disease secondary to CAPS complicated by gastrointestinal symptoms and arthritis which canakinumab could not control. Patients with CAPS who have symptoms that cannot be controlled by canakinumab should be considered for possible co-morbidities.

Peptide Sequence-Dependent Gene Expression of PEGylated Peptide/DNA Complexes.

Oligolysine-based PEG-peptides with 15 or 20 amino acid residues including two cysteines were synthesized to formulate cross-linked polyplex micelles (PMs) incorporating luciferase-coding plasmid DNA (pDNA). Two cysteine residues were separately allocated at the C-terminal, center, or N-terminal of peptide moieties. Although TEM observation showed that all PEG-peptides condensed pDNA into rod-like or toroidal morphologies, the rod length distribution of PMs was affected by both the amino acid sequence and the peptide length of PEG-peptides. In comparison to the cysteine-uninstalled PEG-peptides, the cysteine-installed PEG-peptides exhibited a reductive environment-responsive pDNA release, which was observed in a gel retardation assay. From physicochemical characterizations, a relationship between the amino acid sequence and the in vitro gene expression efficacy of PMs in a cell-free protein synthesis system has been clearly demonstrated. Finally, the cell-based assay using HeLa cells has been tested, and the differences between both results of cell-free and cell-based systems are discussed.

Intravenous abatacept in Japanese patients with polyarticular-course juvenile idiopathic arthritis: results from a phase III open-label study.

BACKGROUND: To investigate efficacy and safety of intravenous abatacept in Japanese patients with active polyarticular-course juvenile idiopathic arthritis (pJIA). METHODS: In this phase III, open-label, multicenter, single-arm study, patients with pJIA aged 4-17 years who failed ≥1 biologic or methotrexate received weight-tiered (< 75 kg: 10 mg/kg; 75-100 kg: 750 mg; > 100 kg: 1000 mg) intravenous abatacept at Weeks 0, 2, 4, and every 4 weeks thereafter. The study comprised a short-term period (16 weeks) and ongoing long-term period. Primary endpoint: Week 16 JIA-American College of Rheumatology criteria 30 (JIA-ACR30) response rate. Secondary endpoints/outcomes included Week 16 JIA-ACR50/70/90 response and inactive disease rates, Childhood Health Assessment Questionnaire-Disability Index (CHAQ-DI), pharmacokinetics, safety, and immunogenicity. Proportions of patients achieving Juvenile Arthritis Disease Activity Score in 27 joints using C-reactive protein (JADAS27-CRP) remission (score < 1) and minimal disease activity (MDA; score < 3.8), were among exploratory endpoints. RESULTS: All 20 patients who received study medication completed the short-term period. During the long-term period, two patients discontinued due to insufficient efficacy or patient decision. Median age and disease duration at baseline were 10.5 and 0.75 years, respectively. Week 16 JIA-ACR30 response rate (primary endpoint) was 90.0% (18/20). JIA-ACR50/70/90 response and inactive disease rates at Week 16 were 75.0% (15/20), 70.0% (14/20), 35.0% (7/20), and 25.0% (5/20), respectively. At Week 52, JIA-ACR30/50/70/90 response and inactive disease rates were observed by 88.9% (16/18), 88.9% (16/18), 83.3% (15/18), 66.7% (12/18) and 44.4% (8/18), respectively. CHAQ-DI improved after Week 12. JADAS27-CRP remission and MDA were achieved by 15.0% (3/20) and 45.0% (9/20) of patients at Week 16, and by 50.0% (9/18) and 78.0% (14/18) of patients at Week 52, respectively. The mean abatacept pre-dose serum concentration was above the target therapeutic exposure (10 µg/ml) from Week 8 through Week 16. All adverse events were of mild/moderate intensity, except for one case of severe gastroenteritis. No deaths, malignancies, or autoimmune disorders were observed. No antidrug antibodies were detected through Week 16; one patient had a positive immunogenic response during the cumulative period. CONCLUSION: Intravenous abatacept was efficacious and well tolerated in Japanese patients with active pJIA. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01835470 . Date of registration: April 19, 2013.

S100A12 and vascular endothelial growth factor can differentiate Blau syndrome and familial Mediterranean fever from systemic juvenile idiopathic arthritis.

OBJECTIVES: Systemic juvenile idiopathic arthritis (sJIA) has recently become regarded as one of the autoinflammatory syndromes (AIS). However, other AIS, such as familial Mediterranean fever (FMF) and Blau syndrome, have been initially misdiagnosed as sJIA because of the clinical similarities. Making the correct diagnosis in the early stage of these AIS is desirable. Therefore, we evaluated serum S100A12 and vascular endothelial growth factor (VEGF) levels to determine if they could be biomarkers for differentiating these AIS. METHOD: Serum S100A12 and VEGF levels were examined in patients with Blau syndrome (n = 4), FMF (n = 4), and sJIA (n = 11) in the active and inactive phases. RESULTS: In the active phase, S100A12 levels were significantly higher in patients with sJIA and FMF compared with those with Blau syndrome (p < 0.001). VEGF levels of patients with sJIA were significantly higher than those of patients with others (p = 0.001). In the inactive phase, there was no significant difference in VEGF levels. However, colchicine-resistant patients or patients without treatment with FMF showed high levels of S100A12 compared with others. CONCLUSIONS: Measuring both serum S100A12 and VEGF levels may be useful for differentiating patients with Blau syndrome and FMF from those with sJIA at the early stage.

Effect of shear stress on structure and function of polyplex micelles from poly(ethylene glycol)-poly(l-lysine) block copolymers as systemic gene delivery carrier.

Structural stability of polyplex micelles (PMs), prepared from plasmid DNA (pDNA) and poly(ethylene glycol)-b-poly(l-lysine) block catiomer (PEG-PLys), was evaluated in terms of their resistance against shear stress. When exposed to shear stress at magnitudes typically present in the blood stream, structural deterioration was observed in PMs owing to the partial removal of PEG-PLys strands. Eventually, impaired PEG coverage of the polyplex core led to accelerated degradation by nucleases, implying that structural deterioration by shear stress in blood stream may be a major cause of rapid clearance of PMs from blood circulation. To address this issue, introduction of disulfide crosslinking into the PM core was shown to be an efficient strategy, which successfully mitigated unfavorable effects of shear stress. Furthermore, improved in vivo blood retention profile and subsequently enhanced antitumor efficacy in systemic treatment of pancreatic adenocarcinoma were confirmed for the crosslinked PMs loaded with pDNA encoding an anti-angiogenic protein, suggesting that high stability under the shear stress during blood circulation may be a critical factor in systemically applicable gene delivery systems.

Prediction of long-term remission of oligo/polyarticular juvenile idiopathic arthritis with S100A12 and vascular endothelial growth factor.

OBJECTIVES: This study aimed to evaluate the usefulness of S100A12 and vascular endothelial growth factor (VEGF) for predicting the stability of remission for discontinuing methotrexate (MTX) and/or biological agents in Japanese patients with oligo/polyarticular juvenile idiopathic arthritis (JIA). METHODS: Forty-four patients with oligo/polyarticular JIA who received MTX with or without biological agents were enrolled. Serum concentration of both S100A12 and VEGF were simultaneously evaluated by ELISA in active and in remission phase determined by activity markers including DAS-28. RESULTS: S100A12 and VEGF were correlated with DAS-28. Of the 22 patients with oligo/polyarticular JIA in clinical remission, 13 patients with low S100A12 and VEGF concentrations could discontinue treatment without relapse over 2 years. However, nine patients without low S100A12 and VEGF concentrations relapsed afterwards, even though they had been in clinical remission. The cut-off levels of S100A12 and VEGF for division into two groups of the maintenance remission and relapse groups were 177 ng/ml and 158 pg/ml, respectively. CONCLUSIONS: S100A12 and VEGF are useful markers for assessing disease activity of oligo/polyarticular JIA in remission phase. These markers should be kept low when clinicians consider tapering or discontinuing treatments in oligo/polyarticular JIA patients.

Interleukin-18 for predicting the development of macrophage activation syndrome in systemic juvenile idiopathic arthritis.

To assess the role of IL-6/IL-18 in the pathogenesis of systemic juvenile idiopathic arthritis (s-JIA) and to investigate the clinical significance of serum IL-18 levels for predicting macrophage activation syndrome (MAS) development, we measured the serum IL-6/IL-18 levels in 76s-JIA patients, including 15 with MAS, and compared them with the clinical features. We identified 2 distinct subsets on the basis of serum IL-6/IL-18 levels. The IL-18-dominant subset had more patients who developed MAS. Serum IL-18 levels during active phase in patients with MAS were significantly higher than those without MAS. The cutoff value of serum IL-18 levels for predicting MAS development was 47750 pg/ml. The patients with IL-18 dominant subset at their disease onset were significantly more likely to develop MAS after TCZ therapy started. IL-18 might have a key role in the pathogenesis of MAS. Serum IL-18 levels >47750 pg/ml might be useful to predict MAS development.

Effect of polymer structure on micelles formed between siRNA and cationic block copolymer comprising thiols and amidines.

Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.

Polyion complex stability and gene silencing efficiency with a siRNA-grafted polymer delivery system.

An siRNA-grafted polymer through disulfide linkage was prepared to improve the physicochemical properties and transfection efficacies of the polyion complexes (PICs) as a nanocarrier of siRNA. The siRNA-grafted polymer formed stable PICs due to its larger numbers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-grafted polymer showed a potent gene silencing effect without cytotoxicity or immunogenicity, demonstrating a promising feature of the siRNA-grafted polymer to construct the PIC-based nanocarrier for in vivo siRNA delivery.

Thermodynamics of DNA condensation induced by poly(ethylene glycol)-block-polylysine through polyion complex micelle formation.

Isothermal titration calorimetry (ITC) was carried out to explore the condensation process of plasmid DNA (pDNA) molecules induced by poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) as a condensing agent. The ITC curves measured can be divided into two distinctive endothermic binding processes: the first was the binding of PEG-PLL to the elongated pDNA, and the second was the binding that accompanied the pDNA conformational transition. The thermodynamic parameters were obtained by fitting each ITC curve using our recently developed fitting method. The binding of PEG-PLL to the pDNA was accompanied by a small increase in enthalpy, a large increase in entropy, and a large decrease in free energy. The binding stabilized as the polymerization degree of PLL on PEG-PLL increased and the salt concentration decreased. Changes in the thermodynamic parameters are discussed in relation to both the polymerization degree of PLL on PEG-PLL and the salt concentration.

Enhanced transfection with silica-coated polyplexes loading plasmid DNA.

Silica-coating of positively charged polyplexes was demonstrated through silicic acid condensation to improve the polyplexes for enhanced complex stability and transfection efficiency. Silicic acid was efficiently condensed by polycations to form a silica network in the polyplex through electrostatic interaction and hydrogen bonding. The silica-coated (SC) polyplexes had an anionic surface charge of -20 mV and were 10-20 nm larger in size compared to the non-silica-coated control (+33.4 mV, 106 nm). Silica-coating significantly improved the polyplex stability against both dissociations by counter polyanion exchange and aggregation by salt. The silica network was dissolved to form silicic acid by removing free silicic acid based on the equilibrium, SiO(2) + 2H(2)O right arrow over left arrow Si(OH)(4). Indeed, dialysis of the SC polyplex solution against excess silica-free buffer permitted plasmid DNA release from the silica-coated polyplex, indicating the reversible nature of the silica-layer. The SC polyplex achieved significantly higher transfection efficiency without serious cytotoxicity compared to the polyplex without silica-coating. Detailed examinations of transfection using SC polyplexes revealed that the enhanced transfection efficiency was because of facilitated endosomal escape, possibly due to the protonation of the silica in acidic endosomal compartments. These findings demonstrate the utility of the silica-coating technique for polyplex-mediated gene delivery.