海藻酸包衣的季铵化壳聚糖纳米粒用于胰岛素口服给药的研究

本研究的目的是开发海藻酸包衣的壳聚糖纳米粒口服递送胰岛素。采用三聚磷酸钠（TPP）离子交联作用将N-[（2-羟基-3-三甲基铵）丙基]壳聚糖氯化物（HTCC）制备得到季铵化壳聚糖纳米粒（HTCC-T纳米粒）,然后在温和搅拌条件下滴加入海藻酸钠溶液,进一步形成海藻酸包衣季铵化壳聚糖纳米粒（HTCC-A纳米粒）。分别采用粒度仪、透射电镜和HPLC分析对HTCC-A纳米粒进行了粒径、zeta电位、表面形态、载药量和包封率的表征。结果表明,HTCC-A纳米粒为均匀的球形颗粒,大小为（322.2±8.5）nm,表面带有正电荷（（14.1±0.6）mV）。体外释放结果表明,在不同p H值的释放介质中,HTCC-A纳米粒的释放行为与HTCC-T纳米粒（未用海藻酸包衣）有很大的不同,这表明海藻酸包衣可以显著改善纳米粒中胰岛素的释放行为。同时,体外酶解试验和圆二色散图谱进一步证实,海藻酸包衣可以显著改善纳米粒中胰岛素结构稳定性。HTCC-A纳米粒十二指肠给药的相对药理生物利用度为8.0%±2.5%。与HTCC-T纳米粒口服给药相比,HTCC-A纳米粒的相对药理生物利用度显著增加（P〈0.05）,是HTCC-T纳米粒的2.2倍。由此可见,海藻酸包衣季铵化壳聚糖纳米粒（HTCC-A纳米粒）将可能成为一种有效的口服递送载体系统用于提高胰岛素的体内口服吸收效果。     

Self-assembly pH-sensitive chitosan/alginate coated polyelectrolyte complexes for oral delivery of insulin

Polyelectrolyte complexes (PEC) provide new opportunities for controlled release system of drugs, and have potentials to address challenges on the way to effective oral insulin delivery. Here, an innovative pH-sensitive PEC for insulin oral administration was developed, which was formed by self-assembly of two oppositely charged nanoparticles (chitosan-coated nanoparticles and alginate-coated nanoparticles) through electrostatic interaction via optimised double emulsion method. The encapsulation efficiency of insulin-loaded alginate-coated and chitosan-coated nanoparticles were 81.5?±?7.4% and 55.2?±?7.0%, respectively, and the particle size of these nanoparticles were in 200–300?nm range. The pH-dependent morphology of PEC was observed by transmission electron microscopy. The PEC exhibited insulin release profile triggered by pH in vitro and was non-cytotoxicity against Caco-2 cell. The insulin-loaded PEC could decrease blood glucose levels effectively and prolong insulin release after oral administration to diabetic rats. The results illustrated that the as-prepared PEC may be employed as a potential oral insulin delivery system.     

In-vitro and in-vivo cytotoxicity and efficacy evaluation of novel glycyl-glycine and alanyl-alanine conjugates of chitosan and trimethyl chitosan nano-particles as carriers for oral insulin delivery

Purpose

The aim of this research work was to explore the possibility of providing multifunctional oral insulin delivery system by conjugating several types of dipeptides on chitosan and trimethyl chitosan to be used as drug carriers.

Nasal delivery of insulin using chitosan microspheres

Nasal delivery of insulin is an alternative route for administration of this drug. The objective of this study was preparation of chitosan microspheres for insulin nasal delivery. After preparation of insulin chitosan microspheres by emulsification-cross linking process, the effect of chitosan quantity (200–400?mg), cross-linker type (ascorbic acid or ascorbyl palmitate) and amount (70–140?mg) were studied on the morphology, particle size, loading efficiency, flow and release of insulin from the microspheres by a factorial design. Optimized formulation was administered nasally in four groups of diabetic rats and their serum insulin levels were analysed by the insulin enzyme immunoassay kit and the serum glucose by the glucose oxidase kits. Insulin loading in microspheres was between 4.7–6.4% w/w, preparation efficiency more than 65% and mean particle size was 20–45?µm. In most cases, drug released followed a Higuchi model. Ascorbic acid caused an increase in stability, particle size and T_50% while decreased the loading efficiency and production efficiency. Increasing the chitosan content, increased particle size, flow and insulin release rate form the microspheres. The increase of cross-linking percentage decreased the flow and size of the microspheres while increase of cross-linking percentage promoted the stability and decreased DE₈% of insulin. Microspheres containing 400?mg of chitosan and 70?mg ascorbyl palmitate caused a 67% reduction of blood glucose compared to i.v. route and absolute bioavaliability of insulin was 44%. The results showed that chitosan microspheres of insulin are absorbable from nasal route.     

Effective insulin delivery using starch nanoparticles as a potential trans-nasal mucoadhesive carrier

Mucoadhesive nanoparticles (NPs) could be an exciting prospect for trans-nasal insulin delivery as they have higher surface area to cover highly vascularised nasal absorptive area providing a greater concentration gradient; hence the present study makes an attempt in this regard. Starch NPs were prepared by different crosslinkers using various methodologies and were loaded with insulin. Emulsion crosslinked particles were smaller in size compared to gel method (351 vs 997 nm), and size is further reduced when epichlorohydrin is used as crosslinking agent compared to POCl3 (194 vs 810 nm). NPs of epichlorohydrin emulsion were further optimized with variable crosslinking to evaluate the effect of degree of crosslinking on in vivo performance. In vitro, a size dependent first order diffusion controlled insulin release with an initial burst effect was found, which is higher with NPs of small size and least crosslinking. Formulation of EE-NPs with Na glycocholate showed a superior hypoglycemic action compared to other NPs formulations containing the former and lysophosphatidylcholine as permeation enhancers. The hypoglycemic effects were more pronounced with medium crosslinked NPs (EE-L2-NPs), which showed a nadir of 70% reduction of plasma glucose and significant effects until 6h. The peak plasma insulin level (Cmax) of medium crosslinked EE-L2-NPs (258 muIU/ml at 1h) vindicates the pharmacodynamic effect, which was found to be superior compared to all other formulations. The release rate and higher associated surface area might work in tandem, and could be greatly amplified when combined with permeation enhancers to make starch NPs an efficient trans-nasal mucoadhesive carrier of insulin.     

Formulation and characterization of an oily-based system for oral delivery of insulin

The present work explored the possibility of formulating an oral insulin delivery system by combining the advantages of nanoencapsulation and the use of oily vehicle. The parameters affecting formulation such as association efficiency were characterized. The preparation was evaluated for its chemical, physical and biological stability. The preparation has unimodal particle size distribution with a mean diameter of 108 ± 9 nm. Insulin was protected from gastric enzymes by incorporation into lipid-based formulation. The results of RP HPLC and ELISA indicated that insulin was able to withstand the preparation procedure. Insulin in the preparations was stable for a period of one month at storage temperatures of 4 and 25 °C. It was also biologically active and stable as demonstrated by the remarkable reduction of blood glucose levels of the STZ-diabetic rats after oral administration of the preparation. Moreover, hypoglycemic effect of nanoparticles administered orally was sustained for a longer period of time compared to the subcutaneous injection. These results clearly evidenced the ability of the nanoparticles to enhance the pharmacological response of insulin when given orally and could be used to deliver other peptides.     

The in vivo fate of nanoparticles and nanoparticle-loaded microcapsules after oral administration in mice: Evaluation of their potential for colon-specific delivery

Anti-cancer drug loaded-nanoparticles (NPs) or encapsulation of NPs in colon-targeted delivery systems shows potential for increasing the local drug concentration in the colon leading to improved treatment of colorectal cancer. To investigate the potential of the NP-based strategies for colon-specific delivery, two formulations, free Eudragit® NPs and enteric-coated NP-loaded chitosan–hypromellose microcapsules (MCs) were fluorescently-labelled and their tissue distribution in mice after oral administration was monitored by multispectral small animal imaging. The free NPs showed a shorter transit time throughout the mouse digestive tract than the MCs, with extensive excretion of NPs in faeces at 5 h. Conversely, the MCs showed complete NP release in the lower region of the mouse small intestine at 8 h post-administration. Overall, the encapsulation of NPs in MCs resulted in a higher colonic NP intensity from 8 h to 24 h post-administration compared to the free NPs, due to a NP ‘guarding’ effect of MCs during their transit along mouse gastrointestinal tract which decreased NP excretion in faeces. These imaging data revealed that this widely-utilised colon-targeting MC formulation lacked site-precision for releasing its NP load in the colon, but the increased residence time of the NPs in the lower gastrointestinal tract suggests that it is still useful for localised release of chemotherapeutics, compared to NP administration alone. In addition, both formulations resided in the stomach of mice at considerable concentrations over 24 h. Thus, adhesion of NP- or MC-based oral delivery systems to gastric mucosa may be problematic for colon-specific delivery of the cargo to the colon and should be carefully investigated for a full evaluation of particulate delivery systems.     

Stealth Amphotericin B nanoparticles for oral drug delivery: In vitro optimization

Purpose

Amphotericin B (AmB) is an effective anti-fungal and anti-leishmanial agent. However, AmB has low oral bioavailability (0.3%) and adverse effects (e.g., nephrotoxicity). The objectives of this study were to improve the oral bioavailability by entrapping AmB in pegylated (PEG) poly lactide co glycolide copolymer (PLGA–PEG) nanoparticles (NPs). The feasibility of different surfactants and stabilizers on the mean particle size (MPS) and entrapment efficiency were also investigated.

Materials and methods

NPs of AmB were prepared by a modified emulsification diffusion method employing a vitamin E derivative as a stabilizer. Physicochemical properties and particle size characterization were evaluated using Fourier Transform Infra-Red spectroscopy (FTIR), differential scanning calorimetry, scanning electron microscopy and transmission electron microscopy. Moreover, in vitro dissolution profiles were performed for all formulated AmB NPs.

Results

MPS of the prepared spherical particles of AmB ranged from 26.4 ± 2.9 to 1068 ± 489.8 nm. An increased stirring rate favored AmB NPs with a smaller MPS. There was a significant reduction in MPS, drug content and drug release, when AmB NPs were prepared using the diblock polymer PLGA–PEG with 15% PEG. Addition of three emulsifying agents poly vinyl pyrrolidone (PVP), Vitamin E (TPGS) and pluronic F-68 to AmB formulations led to a significant reduction in particle size and increase in drug entrapment efficiency (DEE) compared to addition of PVP alone. FTIR spectroscopy demonstrated a successful loading of AmB to pegylated PLGA–PEG copolymers. PLGA–PEG copolymer entrapment efficiency of AmB was increased up to 56.7%, with 92.7% drug yield. After a slow initial release, between 20% and 54% of AmB was released in vitro within 24 h phosphate buffer containing 2% sodium deoxycholate and were best fit Korsmeyer–Peppas model. In conclusion, PLGA–PEG diblock copolymer with 15% PEG produced a significant reduction (>70%) in MPS with highest drug content. The percentage of PEG in the copolymer and the surfactant/stabilizer used had a direct effect on AmB release in vitro, entrapment efficiency and MPS. These developed formulations are feasible, effective and improved alternatives to other carriers for oral delivery of AmB.     

Exploring the potential of functional polymer-lipid hybrid nanoparticles for enhanced oral delivery of paclitaxel

Most biopharmaceutics classification system (BCS) class IV drugs, with poor solubility and inferior permeability, are also substrates of P-glycoprotein (P-gp) and cytochrome P450 (CYP450), leading to their low oral bioavailability. The objective of this study is to explore the potential of using functional polymer-lipid hybrid nanoparticles (PLHNs) to enhance the oral absorption of BCS IV drugs. In this paper, taking paclitaxel (PTX) as a drug model, PTX-loaded PLHNs were prepared by a self-assembly method. Chitosan was selected to modify the PLHN to enhance its mucoadhesion and stability. Three P-gp inhibitors (D-α-tocopherol polyethylene glycol 1000 succinate, pluronic P123 and Solutol^Ⓡ HS15) were incorporated into selected PLHNs, and a CYP450 inhibitor (the extract of VBRB, BC0) was utilized to jointly promote the drug absorption. Properties of all the PLHNs were characterized systemically, including particle size, zeta potential, encapsulation efficiency, morphology, stability, in vitro drug release, mucoadhesion, in situ intestinal permeability and in vivo systemic exposure. It was found mucoadhesion of the CS-modified PLHNs was the strongest among all the formulations tested, with absolute bioavailability 21.95%. P-gp and CYP450 inhibitors incorporation further improved the oral bioavailability of PTX to 42.60%, 8-fold increase compared with that of PTX itself (4.75%). Taken together, our study might shed light on constructing multifunctional PLHNs based on drug delivery barriers for better oral absorption, especially for BCS IV drugs.     

Chitosan phthalate microspheres for oral delivery of insulin: preparation, characterization, and in vitro evaluation

Chitosan phthalate polymer was synthesized and its microspheres were prepared by emulsion phase separation technique. The characterization of microspheres was determined by means of FTIR spectroscopy, electron microscopy, particle size, and zeta potential. The insulin was loaded to the microspheres by passive absorption technique. The peptic and tryptic enzymes degradation of insulin in microspheres was investigated. The in vitro release behavior of the microspheres was investigated under different pH conditions (pH 2.0 and pH 7.4). The degree of phthalate substitution in the synthesized polymer was 20%. The prepared microspheres were spherical with an average diameter 46.34 μ m. The insulin-loading capacity was 62%. Chitosan phthalate microspheres protect the insulin from gastric enzymes degradation that may enhance the oral stability of insulin. The encapsulated insulin was quickly released in a phosphate buffer saline (pH 7.4), whereas a small amount of insulin was released under acidic condition (0.1N HCl; pH 2.0) because under acidic conditions, carboxylic groups present in the system exist in nonionized form and are poorly hydrophilic. However, in alkaline conditions, it exists in ionized form and is considerably hydrophilic. The results suggest that chitosan phthalate microspheres may be used as a potential carrier for oral insulin delivery.     

Application of polymeric nanoparticles and micelles in insulin oral delivery

Diabetes mellitus is an endocrine disease in which the pancreas does not produce sufficient insulin or the body cannot effectively use the insulin it produces. Insulin therapy has been the best choice for the clinical management of diabetes mellitus. The current insulin therapy is via subcutaneous injection, which often fails to mimic the glucose homeostasis that occurs in normal individuals. This provokes numerous attempts to develop a safe and effective noninvasive route for insulin delivery. Oral delivery is the most convenient administration route. However, insulin cannot be well absorbed orally because of its rapid enzymatic degradation in the gastrointestinal tract. Therefore, nanoparticulate carriers such as polymeric nanoparticles and micelles are employed for the oral delivery of insulin. These nanocarriers protect insulin from degradation and facilitate insulin uptake via a transcellular and/or paracellular pathway. This review article focuses on the application of nanoparticles and micelles in insulin oral delivery. The recent advances in this topic are also reviewed.     

Tocol modified glycol chitosan for the oral delivery of poorly soluble drugs

The aim of this study was to develop tocol derivatives of chitosan able (i) to self-assemble in the gastrointestinal tract and (ii) to enhance the solubility of poorly soluble drugs. Among the derivatives synthesized, tocopherol succinate glycol chitosan (GC-TOS) conjugates spontaneously formed micelles in aqueous solution with a critical micelle concentration of 2 μg mL⁻¹. AFM and TEM analysis showed that spherical micelles were formed. The GC-TOS increased water solubility of 2 model class II drugs. GC-TOS loading efficiency was 2.4% (w/w) for ketoconazole and 0.14% (w/w) for itraconazole, respectively. GC-TOS was non-cytotoxic at concentrations up to 10 mg mL⁻¹. A 3.4-fold increase of the apparent permeation coefficient of ketoconazole across a Caco-2 cell monolayer was demonstrated. Tocol polymer conjugates may be promising vehicles for the oral delivery of poorly soluble drugs.     

pH-sensitive polymers for cytoplasmic drug delivery

The pH-sensitive drug delivery systems could be triggered by a mild acidic environment, such as that occurring in solid tumors, inflammatory tissues and intracellular endosomal compartments. Moreover, the cytoplasmic delivery of internalized macromolecules (such as oligonucleic acid, siRNA, DNA, proteins and polymer–antibody complex) will be possible. Synthetic polymers – such as polyanions (acrylic acid derivatives) and polycations (poly ethylenimine and chitosan complexes) – are among the most popular compounds studied for intracellular trafficking of drugs. As research is progressing in the area of cytoplasmic delivery, many novel and innovative applications making use of the unique properties of pH-sensitive polymers are expected in the future.     

Antacid co-encapsulated polyester nanoparticles for peroral delivery of insulin: Development,pharmacokinetics, biodistribution and pharmacodynamics

The in vitro/in vivo characterization of antacid-insulin co-encapsulated poly(lactide-co-glycolide) (PLGA) nanoparticles is presented here. The optimized nanoparticle composition has 1% surfactant (didodecyl dimethylammonium bromide) and 2% antacid (magnesium hydroxide or zinc carbonate) in the size range ∼136–143 nm with ∼81–85% entrapment of insulin at a 4% (w/w) initial load to that of polymer. Molecular characterization using circular dichroism, fluorescence and Fourier transform infrared spectroscopy showed that the structural integrity of insulin was maintained during formulation. Furthermore, the encapsulated insulin was well protected under in vitro simulated gastric and intestinal fluids. Nanoparticle insulin results in six fold increase in oral bioavailability to that of plain insulin in healthy rats. In diabetic rats, a 120 IU/kg oral dose of insulin nanoparticles achieved an equivalent blood glucose lowering effect to a 20 IU/kg subcutaneous (sc) dose of insulin solution, the nadir in blood glucose concentration occurring 24 h and 1 h post-administration, respectively. Both sc insulin and oral nanoparticle insulin partially attenuated hyperglycemia-induced inflammation caused by tumor necrosis factor α, but not by interleukin-6 or C-reactive protein; on the other hand, subcutaneous insulin was found to be more effective on lipid profile measured in the form of high density lipoprotein, cholesterol and triglyceride. Successful oral insulin could be beneficial in type II complications.     

Effect of pH-sensitive nanoparticles on inhibiting oral biofilms

Dental caries is a biofilm-related preventable infectious disease caused by interactions between the oral bacteria and the host’s dietary sugars. As the microenvironments in cariogenic biofilms are often acidic, pH-sensitive drug delivery systems have become innovative materials for dental caries prevention in recent years. In the present study, poly(DMAEMA-co-HEMA) was used as a pH-sensitive carrier to synthesize a chlorhexidine (CHX)-loaded nanomaterial (p(DH)@CHX). In vitro, p(DH)@CHX exhibited good pH sensitivity and a sustained and high CHX release rate in the acidic environment. It also exhibited lower cytotoxicity against human oral keratinocytes (HOKs) compared to free CHX. Besides, compared with free CHX, p(DH)@CHX showed the same antibacterial effects on S. mutans biofilms. In addition, it had no effect on eradicating healthy saliva-derived biofilm, while free CHX exhibited an inhibitory effect. Furthermore, the 16s rDNA sequencing results showed that p(DH)@CHX had the potential to alter oral microbiota composition and possibly reduce caries risk. In conclusion, the present study presents an alternative option to design an intelligent material to prevent and treat dental caries.     

Preparation and in vitro evaluation of bFGF-loaded chitosan nanoparticles

The objective of our study was to prepare and characterize basic fibroblast growth factor (bFGF)-loaded nanoparticles. Protein-loaded chitosan nanoparticles were obtained by ionotropic gelation process based on the interaction between chitosan and tripolyphosphate (TPP). The protein-loading capacity and encapsulation efficiency were 0.021% and 27.388%, respectively. The bFGF-loaded nanoparticles have a mean diameter of 424 nm, a narrow size distribution, spherical shape and positive surface charges. In vitro release showed that the extent of release was 68% at 24 hr. The protein integrity was investigated by SDS-PAGE analysis that confirmed protein integrity was not affected by the encapsulation procedure and release conditions.     

The upregulated intestinal folate transporters direct the uptake of ligand-modified nanoparticles for enhanced oral insulin delivery

Transporters are traditionally considered to transport small molecules rather than large-sized nanoparticles due to their small pores. In this study, we demonstrate that the upregulated intestinal transporter (PCFT), which reaches a maximum of 12.3-fold expression in the intestinal epithelial cells of diabetic rats, mediates the uptake of the folic acid-grafted nanoparticles (FNP). Specifically, the upregulated PCFT could exert its function to mediate the endocytosis of FNP and efficiently stimulate the traverse of FNP across enterocytes by the lysosome-evading pathway, Golgi-targeting pathway and basolateral exocytosis, featuring a high oral insulin bioavailability of 14.4% in the diabetic rats. Conversely, in cells with relatively low PCFT expression, the positive surface charge contributes to the cellular uptake of FNP, and FNP are mainly degraded in the lysosomes. Overall, we emphasize that the upregulated intestinal transporters could direct the uptake of ligand-modified nanoparticles by mediating the endocytosis and intracellular trafficking of ligand-modified nanoparticles via the transporter-mediated pathway. This study may also theoretically provide insightful guidelines for the rational design of transporter-targeted nanoparticles to achieve efficient drug delivery in diverse diseases.     

Development and in vivo evaluation of chitosan nanoparticles for the oral delivery of albumin

Albumin is used as a plasma expander in critically ill patients and for several other clinical applications mainly via intravenous infusion. Oral administration of albumin can improve patient compliance although limited oral bioavailability of proteins is still a major challenge. Although nanomaterials have been extensively utilized for improving oral delivery of proteins, albumin has been utilized only as either a model drug or as a carrier for drug delivery. In the current study, for the first time, chitosan nanoparticles have been developed and extensively optimized to improve oral bioavailability of albumin as a therapeutic protein. Several characterizations have been performed for the albumin-loaded nanoparticles (e.g. drug encapsulation efficiency, DSC, FTIR, particle size, zeta potential, morphology, release kinetics, and enzymatic stability). Nanosized spherical particles were prepared and demonstrated high stability over three months either in a powdered form or as suspensions. Sustained release of albumin over time and high enzymatic stability as compared to the free albumin were observed. In vivo, higher serum concentrations of albumin in normal rabbits and cirrhotic rats were attained following oral and intraperitoneal administrations of the albumin-loaded nanoparticles as compared to the free albumin. The nanoparticles developed in the current study might provide efficient nanovehicles for oral administration of therapeutic albumin.     

Microencapsulated chitosan nanoparticles for lung protein delivery

It has already been demonstrated that spray drying is a very valuable technique for producing dry powders adequate for pulmonary delivery of drugs. We have developed chitosan/tripolyphosphate nanoparticles that promote peptide absorption across mucosal surfaces. The aim of this work was to microencapsulate protein-loaded chitosan nanoparticles using typical aerosol excipients, such as mannitol and lactose, producing microspheres as carriers of protein-loaded nanoparticles to the lung. The results showed that the obtained microspheres are mostly spherical and possess appropriate aerodynamic properties for pulmonary delivery (aerodynamic diameters between 2 and 3 μm, apparent density lower than 0.45 g/cm³). Moreover, microspheres morphology was strongly affected by the content of chitosan nanoparticles. These nanoparticles show a good protein loading capacity (65–80%), providing the release of 75–80% insulin within 15 min, and can be easily recovered from microspheres after contact with an aqueous medium with no significant changes in their size and zeta potential values. Therefore, this work demonstrated that protein-loaded nanoparticles could be successfully incorporated into microspheres with adequate characteristics to reach the deep lung, which after contact with its aqueous environment are expected to be able to release the nanoparticles, and thus, the therapeutic macromolecule.     

Cholic acid modified N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride for superoxide dismutase delivery

A series of novel amphiphilic chitosan derivatives, cholic acid modified N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride (HTCC-CA) with different quaternization degrees and cholic acid substitutions were synthesized in this study. HTCC-CA is biocompatible and forms particles in aqueous solution. The binding with superoxide dismutase (SOD) at pH 6.8 destroys the original aggregates of HTCC-CA and produces smaller SOD/HTCC-CA complex nanoparticles via electrostatic and hydrophobic interactions. The SOD loading efficiency and loading capacity of HTCC-CA can reach to more than 90% and 45%, respectively. Confocal laser scanning microscopy observation and flow cytometry analysis reveal that SOD/HTCC-CA complex nanoparticles greatly enhance the cellular internalization of the loaded SOD. The SOD activities and malonaldehyde concentrations in the serum and organs of the rats, administrated intravenously with free SOD, free HTCC-CA, and SOD/HTCC-CA nanoparticles, were assayed to evaluate the antioxidant efficiency in vivo. The results demonstrate that free HTCC-CA is effective to scavenge superoxide radicals in the blood circulation and SOD/HTCC-CA nanoparticles have better antioxidant efficiency than free SOD as well as free HTCC-CA.