Efficient delivery of the Bcl-2 antisense oligonucleotide G3139 via nucleus-targeted aCD33-NKSN nanoparticles.

G3139 is an antisense oligodeoxyribonucleotide (ODN) developed as a Bcl-2 down-regulating agent for the treatment of acute myelogenous leukemia (AML). However, the clinical efficacy of G3139 has been shown to be limited due to its rapid plasma clearance and low permeability. To enhance the effective delivery of G3139, this work prepared a novel nano gene delivery vector (aCD33-NKSN) consisting of a CD33 antigbody (aCD33), a nuclear localization signal (NLS), gene fusion peptides (KALA), and stearic acid (SA) for CD33 antigen targeting and nuclear localization. The aCD33-NKSN/G3139 nanoparticles were spherical and uniformly sized with a positive charge and sustained release. They had an excellent G3139 loading capacity and colloidal stability. The aCD33-NKSN/G3139 delivered G3139 into the nucleus of Kasumi-1 cells and aCD33-NKSN/G3139 could more effectively inhibited Bcl-2 expression and induced apoptosis in Kasumi-1 cells versus free G3139. The aCD33-NKSN/G3139 administration was more effective at inhibiting tumor growth, and significantly prolonged the survival time of mice in contrast to free G3139. The results illustrate that aCD33-NKSN/G3139 nanoparticles could improve the antitumor activity of encapsulated G3139 due to aCD33 targeting and the ability to perform nuclear localization, The results offer a promising clinical application potential for the treatment of acute myeloid leukemia.

Design of a Novel Nucleus-Targeted NLS-KALA-SA Nanocarrier to Delivery Poorly Water-Soluble Anti-Tumor Drug for Lung Cancer Treatment.

In this study, we designed a novel nucleus-targeted nanocarrier (NLS-KALA-SA, NKSN) consisting of Kala peptide (KALA), nuclear localization signal (NLS) and stearic acid (SA) using Fmoc solid phase synthesis method. We chose Curcumin (CUR), Paclitaxel (PTX), Ginsenoside compound K(CK) as models of poorly water-soluble antitumor drugs, The drugs loaded NLS-KALA-SA nanoparticles (CUR/NKSN, PTX/NKSN, CK/NKSN) were obained by the dialysis method, their physicochemical properties were determined and antitumor activity were evaluated. The NLS-KALA-SA nanoparticles were spherical shaped with an average size of 76.4 ± 7.6 mm and a zeta potential of 43.7 ± 5.8 mV. The drug-loaded NLS-KALA-SA nanoparticles were above 86.1% and 17.1% in entrapment efficiency and drug loading capacity, and had sustained drug release behavior. Biodistribution and cellular uptake study exhibited that PTX/NKSN mainly distributed in tumor site of A549-bearing mice, and coumarin-6(C6) loaded NLS-KALA-SA nanoparticle (C6/NKSN) was predominantly accumulated in the nucleus of A549 cells. Western blot analysis indicated that PTX/NKSN could more remarkably inhibit Bcl-2 expression and enhance the expression of Bax and Caspase-3 as compared to the controls in A549 cells. Cell apoptosis and antitumor activity study showed that PXT/NKSN could more obviously induce apoptosis of A549 cells compared with free PXT, the PTX/NKSN administration was more effective than free PTX for lung cancer treatment and displayed mild toxicity in A549-bearing mice. The results demonstrates that the NLS-KALA-SA nanoparticles system could enhance the antitumor effects of the encapsulated drug and reduce tissue toxicity due to its long circulating properties and tumor targeting, which might provide a promising strategy for lung cancer treatment.

Photoluminescent functionalized carbon quantum dots loaded electroactive Silk fibroin/PLA nanofibrous bioactive scaffolds for cardiac tissue engineering.

Tissue engineering and stem cell rehabilitation are the hopeful aspects that are being investigated for the management of Myocardial Infarction (MI); cardiac patches have been used to start myocardial rejuvenation. In this study, we engineered p-phenylenediamine surface functionalized (modif-CQD) into the Silk fibroin/PLA (SF/PLA) nanofibrous bioactive scaffolds with improved physico-chemical abilities, mechanical and cytocompatibility to cardiomyocytes. The micrograph results visualized the morphological improved spherical modif-CQD have been equivalently spread throughout the SF/PLA bioactive cardiac scaffolds. The fabricated CQD@SF/PLA nanofibrous bioactive scaffolds were highly porous with fully consistent pores; effectively improved young modulus and swelling asset for the suitability and effective implantation efficacy. The scaffolds were prepared with rat cardiomyocytes and cultured for up to 7 days, without electrical incentive. After 7 days of culture, the scaffold pores all over the construct volume were overflowing with cardiomyocytes. The metabolic activity and viability of the cardiomyocytes in CQD@SF/PLA scaffolds were significantly higher than cardiomyocytes in Silk fibroin /PLA scaffolds. The integration of CQD also influenced greatly and increases the expression of cardiac-marker genes. The results of the present investigations evidently recommended that well-organized cardiac nanofibrous scaffold with greater cardiac related mechanical abilities and biocompatibilities for cardiac tissue engineering and nursing care applications.

Caproyl-Modified G2 PAMAM Dendrimer (G2-AC) Nanocomplexes Increases the Pulmonary Absorption of Insulin.

We aimed to investigate the absorption-enhancing effect (AEE) of caproyl-modified G2 PAMAM dendrimer (G2-AC) on peptide and protein drugs via the pulmonary route. In this study, G2 PAMAM dendrimer conjugates modified with caproic acid was synthesized, the pulmonary absorption of insulin as models with or without G2-AC were evaluated. The results indicated that G2-AC6 exhibited a greatest AEE for insulin in various caproylation levels of G2-AC. G2-AC6 could significantly enhance the absorption of insulin, and the AEE of G2-AC6 was concentration-dependent. In toxicity tests, G2-AC6 displayed no measurable cytotoxicity to the pulmonary membranes over a concentration range from 0.1% (w/v) to 1.0% (w/v). Measurements of the TEER and permeability showed that G2-AC6 significantly reduced the TEER value of CF and increased its Papp value. The results suggested that G2-AC6 could cross epithelial cells by means of a combination of paracellular and transcellular pathways. These findings suggested G2-AC6 at lower concentrations (below 1.0%, w/v) might be promising absorption enhancers for increasing the pulmonary absorption of peptide and protein drugs.

Age-related and gender-stratified differences in the association between high triglyceride and risk of hyperuricemia.

BACKGROUND: Elevated serum uric acid is commonly associated with high triglyceride. However, the relation of triglyceride and hyperuricemia in different gender and age groups is currently not well understood. This study aimed to evaluate age- and gender-related association of high triglyceride with hyperuricemia in a subgroup of Chinese population. METHODS: We retrospectively analyzed physical examination data of 24,438 subjects (12,557 men and 11,881 women) in Kaifeng, China. The alanine aminotransferase, γ-glutamyl transpeptidase, serum creatinine, blood urea nitrogen, total cholesterol, high-density lipoprotein cholesterol, triglyceride and serum uric acid were measured in all subjects. The triglyceride was categorized into < 1.21, 1.21 ~, 1.7 ~, 2.83 ~ and >  5.6 mmol/L subgroups, and odds ratio (OR) and 95% confidence interval (CI) of hyperuricemia were calculated by logistic regression analysis. RESULTS: Univariate and age-adjusted analyses showed that high triglyceride was positively associated with hyperuricemia (p <  0.01). Further age-stratified analysis showed that the positive association was significant in the 20 ~, 30 ~, 40 ~, 50 ~, 60 ~ and 80 ~ age groups in men. In women, no statistically significant was found in 60 ~ and 70 ~ age groups. CONCLUSION: High triglyceride is positively associated with hyperuricemia in both men and women, and this association is age-related, especially in women.

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

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

The inhibiting role of hydroxypropylmethylcellulose acetate succinate on piperine crystallization to enhance its dissolution from its amorphous solid dispersion and permeability.

The purpose of this study was to demonstrate that inhibiting crystallization by HPMCAS played a key role in enhancing dissolution and absorption of piperine (Pip) from its amorphous solid dispersion (ASD). Nucleation induction time and supersaturation tests were used to evaluate the ability of the polymers to inhibit crystallization of Pip. The prepared solid dispersions were characterized by DSC and FTIR. The dissolution rate of Pip from its ASDs was assayed by a dissolution test. Pip permeability was investigated by single-pass intestinal perfusion studies. The order of the ability of polymers to inhibit Pip crystallization was HF > MF > LF > L100-55. The best inhibition effect of HF can be attributed to its hydrophobicity and steric hindrance. Pip is amorphous in polymer matrices when the ratio of Pip/HPMCAS is lower than 1 : 1 and Pip/L100-55 is lower than 3 : 1. IR spectra show that there are hydrogen bonds between the amide groups of Pip and the carboxyl groups of polymer. The order of the ability of polymers to enhance Pip dissolution is HF > MF > LF > L100-55, which coincided with the ability of polymers to inhibit Pip crystallization. Increased apparent permeability via HF-induced supersaturation and decreased apparent permeability via solubilization with L100-55 are demonstrated. Nucleation induction time and supersaturation tests may be used to screen appropriate polymers for preparing ASDs.

LncRNA ENST00113 promotes proliferation, survival, and migration by activating PI3K/Akt/mTOR signaling pathway in atherosclerosis.

BACKGROUND: Atherosclerosis is one of the most common cardiovascular disorders. The dysfunction of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are 2 key factors in the formation of atherosclerosis. This study aims to find strategies to prevent VSMCs and ECs dysfunction for the treatment of atherosclerosis. METHODS: We investigated the expression patterns of long noncoding RNAs (lncRNAs) in 2 pairs of serum samples from both atherosclerosis patients and healthy volunteers through microarray analysis. Then we selected the most up-regulated lncRNA ENAST00113 (lnc00113) to further verify its roles in atherosclerosis. VSMCs, and human umbilical vein endothelial cells (HUVECs) transfected with small interfering RNA (siRNAs) (si-00113-1, si-00113-1) and a negative control (si-NC) were cultured. MTT assay, Caspase 3 enzyme-linked immunosorbent assay (ELISA) assay, and wound healing assay were performed to evaluate whether lnc00113 had an effect on proliferation, apoptosis, and migration ability. Further, the correlation between lnc00113 and PI3K/Akt/mTOR signaling pathway was explored. RESULTS: Microarray results indicated that 243 lncRNAs were up-regulated and 187 lncRNAs were down-regulated. Therefore, we chose the most up-regulated lncRNA ENST (lnc00113) to further explore its roles in atherosclerosis. Real-time polymerase chain reaction (RT-qPCR) results showed that the expression of lnc00113 was highly increased in atherosclerosis patients. In vitro experiment demonstrated that lnc00113 down-regulation significantly suppressed VSMCs and HUVECs proliferation, survival, and migration. Furthermore, we found that the protein expressions of phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt), phosphorylated-mTOR (p-mTOR), and bcl-2 in HUVECs cells transfected with si-00113-1 or si-00113-2 were dramatically decreased compared with si-NC-transfected cells and control cells. However, the total- PI3K (t-PI3K), total-Akt (t-Akt), and total-mTOR (t-mTOR) protein expressions were not changed, indicating that lnc00113 could activate PI3K/Akt/mTOR signaling pathway in atherosclerosis. CONCLUSIONS: This finding identified an important role of lnc00113 in VSMCs and HUVECs that promotes cell proliferation, survival, and migration by activating PI3K/Akt/mTOR signaling pathway, which could probably serve as a promising therapeutic target for atherosclerosis.

Tat-Tagged and Folate-Modified N-Succinyl-chitosan (Tat-Suc-FA) Self-assembly Nanoparticle for Therapeutic Delivery OGX-011 to A549 Cells.

The objective of this study was to develop a novel type of an antisense oligonucleotide (OGX-011) loaded Tat-tagged and folate-modified N-succinyl-chitosan (Tat-Suc-FA) nanoparticles (NPs) for improving tumor targetability. In this study, Tat-Suc-FA/OGX-011NPs were prepared and its physicochemical characterizations were also evaluated. The nanoparticles showed an average diameter of 73 ± 16.6 nm, the zeta potential of +23.6 ± 0.3 mV, and a high entrapment efficiency of 89.6 ± 6.6%. Transmission electron microscopy analysis showed the nanoparticles were mostly spherical and well dispersed. The delivery efficiency of this system was investigated both in vitro and in vivo. In comparison with nontargeted Lipofectamin2000/OGX-011 and free OGX-011, Tat-Suc-FA/GOX-011 showed the highest apoptosis rate of 14.2% ± 1.8% and significant uptake in A549 cells. Tat-Suc-FA NPs loaded with GOX-011 induced significant down-regulation of s-CLU mRNA and protein levels in A549 cells. In A549 tumor-bearing mice model, Tat-Suc-FA/GOX-011 produced a more efficient down-regulation of s-CLU compared to Lipofectamin2000/OGX-011. Furthermore, the combined use of Tat-Suc-FA/OGX-011 with DDP chemotherapy showed a most significant inhibition of tumor growth and greatly enhanced the survival rate of A549 tumor-bearing mice. These findings suggested successful application of Tat-Suc-FA NPs for the high efficiency and specificity in therapeutic delivery of OGX-011 to A549 cells.

Improved intestinal absorption of water-soluble drugs by acetylation of G2 PAMAM dendrimer nanocomplexes in rat.

In search of an effective and less toxic absorption enhancer, we synthesized primary amine acetylation of generation 2 polyamidoamine (G2 PAMAM) dendrimer (Ac-G2) by the reaction of G2 PAMAM dendrimer with acetic anhydride, and evaluated the effects of Ac-G2 on the intestinal absorption of poorly absorbable water-soluble drugs using an in situ closed-loop method in rats. The results indicated that Ac50-G2 had a greatest absorption enhancing effect for 5(6)-carboxyfluorescein (CF) in various acetylation levels of G2 PAMAM dendrimers. Ac50-G2 with various concentrations (0.1-1.0%, w/v) could significantly improve the intestinal absorption of alendronate, CF, and fluorescein isothiocyanate-labeled dextrans (FD4), although they did not enhance the absorption of macromolecular drug of FD10, and the absorption enhancement effect of Ac50-G2 was concentration-dependent. Furthermore, we examined the intestinal membrane damage with or without Ac50-G2. The results displayed Ac50-G2 at lower concentrations (0.1-0.5%, w/v) did not cause any observed toxic effect to the intestinal membranes. These findings suggested Ac50-G2 at lower concentrations (below 0.5%, w/v) might be promising as an effective and safe absorption enhancers to promote the intestinal absorption of poorly absorbable drugs.

Lipid Nanoparticles Loaded with an Antisense Oligonucleotide Gapmer Against Bcl-2 for Treatment of Lung Cancer.

PURPOSE: Bcl-2 is an anti-apoptotic gene that is frequently overexpressed in human cancers. G3139 is an antisense oligonucleotide against bcl-2 that has shown limited efficacy in clinical trials. Here, we report the synthesis of a new antisense oligonucleotide containing additional chemical modifications and its delivery using nanoparticles. METHODS: An oligonucleotide G3139-GAP was synthesized, which has 2'-O-methyl nucleotides at the 5' and 3' ends based on a "gapmer" design. Furthermore, G3139-GAP was incorporated into lipid nanoparticles (LNPs) composed of DOTAP/egg PC/cholesterol/Tween 80. The LNP-loaded G3139-GAP was evaluated in A549 lung cancer cells both in vitro and in a murine xenograft model for biological activity and therapeutic efficacy. RESULTS: The LNPs showed excellent colloidal and serum stability, and high encapsulation efficiency for G3139-GAP. They have a mean particle diameter and zeta potential of 134 nm and 9.59 mV, respectively. G3139-GAP-LNPs efficiently downregulated bcl-2 expression in A549 cells, as shown by 40% and 83% reduction in mRNA and protein levels, respectively. Furthermore, G3139-GAP-LNPs were shown to inhibit tumor growth, prolong survival, and downregulate tumor bcl-2 expression in an A549 murine xenograft tumor model. These data indicate that G3139-GAP-LNPs have excellent anti-tumor efficacy and warrant further evaluation.

Circulating miR-122-5p as a potential novel biomarker for diagnosis of acute myocardial infarction.

BACKGROUND: MicroRNAs (miRNAs) play key roles in cardiac development, and the expression of miRNAs is altered in the diseased heart. The aim of this study was to explore the value of circulating microRNA-122-5p (miR-122-5p) as a potential biomarker for acute myocardial infarction (AMI). METHODS: Plasma samples from 50 patients with AMI and 39 healthy adults (non-AMI controls) were collected. The abundance of circulating miR-122-5p was measured using quantitative real-time PCR (qRT-PCR). The cTnI concentrations of these samples were analyzed by ELISA. RESULTS: Our findings revealed that circulating miR-122-5p expression were increased in AMI patients at 4 h, 8 h, 12 h, and 24 h by contrast to those non-AMI controls and displayed similar trends to that of cTnI concentrations in AMI patients. Further study showed that there is a high correlation between circulating miR-122-5p and cTnI concentrations. At last, the receiver operating characteristic (ROC) curve was performed and showed that circulating miR-122-5p had considerable diagnostic accuracy for AMI with an area under curve (AUC) of 0.855. CONCLUSIONS: Our results implied that circulating miR-122-5p could be a potential biomarker for AMI.

Synthesis of Tat tagged and folate modified N-succinyl-chitosan self-assembly nanoparticles as a novel gene vector.

The purpose of this research was to prepare a novel type of Tat tagged and folate modified N-succinyl-chitosan (Tat-Suc-FA) self-assembly nanoparticles, to provide a new vector for tumor gene therapy. In this study, Tat-Suc-FA polymers was synthesized and characterized using (1)H NMR and FT-IR. The copolymer had a mean diameter of 65 ± 22.6 nm, a zeta potential of 40 ± 0.2 mV. The cytotoxicity assay showed that Tat-Suc-FA polymers were less toxic than chitosan in the tested concentration range (from 2 to 500 µg/ml). Tat-Suc-FA/DNA complexes at various weight ratios were formulated and characterized. Particle sizes of Tat-Suc-FA/DNA complexes were between 54 and 106 nm as determined by dynamic light scattering. Accordingly, Transmission electron microscope photo of Tat-Suc-FA/DNA complexes exhibited a spherical and compact morphology. Zeta potentials of these complexes changed as the weight ratio varied (from 3 to 44 mV). Agarose gel electrophoresis assay showed that Tat-Suc-FA could efficiently condense the DNA, when the weight ratio was above 1.5/1. Together, these results suggest that the low toxic Tat-Suc-FA cationic polymers could be considered for use as a novel type of gene delivery vectors.

Improved tumor targetability of Tat-conjugated PAMAM dendrimers as a novel nanosized anti-tumor drug carrier.

The generation 4-poly-amidoamine-dendrimers (PAMAM G4 dendrimer, P) was conjugated to Tat peptide (Tat, T), a cell-penetrating peptide, in search of an efficient anti-tumor drug delivery vehicle for cancer therapy. In this study, we synthesized BODIPY-labeled Tat-Conjugated PAMAM dendrimers (BPTs) as a novel nanosized anticancer drug carriers and systemically investigated their biodistribution and the tumor accumulation in Sarcoma 180-bearing mice. In addition, the uptake and the cytotoxicity to S180 cells of BPTs thereof were evaluated. The unmodified dendrimer (BP) showed a soon clearance from the blood stream and nonspecific accumulation in tumor. In contrast, the Tat-modified dendrimer, BPT(64) with appropriate particle size showed a better retention in blood and could be accumulated effectively in tumor tissue via the enhanced permeability and retention (EPR) effect. Moreover, BPTs with a high Tat modification rate was accumulated more effectively in tumor tissue. In vitro experiments, these BPTs displayed low cytotoxicity on S180 cells and high uptake to S180 cells. These findings indicate that the nanoparticulate system on the basis of Tat-conjugated PAMAM dendrimers is safer and effective in the concentration range (below 20 µg/ml) to be used as a carrier of anti-tumor drugs for tumor targeting by intravenous administration.

In vivo biodistribution for tumor targeting of 5-fluorouracil (5-FU) loaded N-succinyl-chitosan (Suc-Chi) nanoparticles.

5-Fluorouracil-loaded N-succinyl-chitosan nanoparticles (5-FU-Suc-Chi/NP) were prepared by emulsification solvent diffusion. Biodistribution and tumor targeting were evaluated after i.v. administration of 5-Fu-Suc-Chi/NPs in Sarcoma 180-bearing mice. Also, pharmacokinetic profiles were evaluated after intravenous injection of 5-Fu-Suc-Chi/NP via the tail vein to rats. Our experimental results showed the 5-FU-Suc-Chi/NPs could be sustained at a high level in the blood for a very long time, implying its long systemic retention in the circulation. 5-FU-Suc-Chi/NPs were distributed mainly in tumors and liver, with small quantities being found in kidney and spleen. 5-FU-Suc-Chi/NPs accumulated only slightly in the heart and lung, and lowered the toxic effect of 5-FU in the heart and lung. Pharmacokinetic analysis in plasma showed the area under plasma concentration-time curve (AUC), elimination half-life (t(1/2)), and residence time (MRT) were increased 2.5-fold, 10.98-fold, and 10.8-fold for 5-FU-Suc-Chi/NP compared with that of free 5-FU, respectively. These results indicate that a long half-life in the circulation and tumor targeting of 5-FU-Suc-Chi/NPs are possible.

Nanoparticles of 5-fluorouracil (5-FU) loaded N-succinyl-chitosan (Suc-Chi) for cancer chemotherapy: preparation, characterization--in-vitro drug release and anti-tumour activity.

N-Succinyl-chitosan has favourable properties as a drug carrier, such as biocompatibility, low toxicity and long-term retention in the body. It is a good candidate for cancer chemotherapy as a polymeric drug carrier. This study describes the preparation and characterization of 5-fluorouracil-loaded N-succinyl-chitosan nanoparticles (5-FU-Suc-Chi/NP) by an emulsification solvent diffusion method. The influence of the initial 5-FU concentration on the nanoparticle characteristics and release behaviour in phosphate-buffered saline solution (PBS) was evaluated. The Suc-Chi nanoparticles had a particle diameter (Z-average) in the range 202 approximately 273 nm and a negative zeta-potential (approx. -27 to -18 mV). The formulation with an initial 5-FU concentration of 1000 microg mL-1 provided the highest loading capacity (19%) and the highest extent of release (61% at 24 h). The 5-FU-Suc-Chi/NP showed good anti-tumour activity against Sarcoma 180 solid tumour and mild toxicity. According to the data obtained, this Suc-Chi-based nanotechnology opens new and interesting perspectives for cancer chemotherapy.

Preparation of N-succinyl-chitosan and its physical-chemical properties as a novel excipient.

The aim of this work is to prepare N-succinyl-chitosan (Suc-Chi) and measure physical-chemical properties for Suc-Chi as excipients. Suc-Chi were prepared via ring-opening reactions with succinic anhydride in Dimethyl Sulfoxide system. The physical-chemical properties of Suc-Chi, such as the degree of substitution (DS), solubility, isoelectric point (pI), glass transition temperature (Tg), partition coefficient (P(app)) and zata potential were detected respectively in order to evaluate their possibility as drug carriers. We obtained Suc-Chi DAC-90 (DS=0.33) and the data of physical-chemical properties for the product. The knowledges of physical-chemical properties for Suc-Chi are valuable for basic or applied purposes in biomedical and pharmaceutical sciences stabilization.