Microbeads mediated oral plasmid DNA delivery using polymethacrylate vectors: an effectual groundwork for colorectal cancer

Abstract

This study was aimed to develop and evaluate p53 polyplex-loaded enteric-coated calcium pectinate microbeads for oral gene delivery as an effective novel alternative for colorectal cancer therapy. Mutation in p53 is the key event in colorectal cancer (CRC) and an important target for the treatment of CRC through gene therapy. Polymethacrylates-based non-viral vectors were evaluated for their ability to complex, protect and transfect p53 (wt) into colon cancer cell line. Polyplexes were formulated by complexation of cationic polymer with anionic pDNA at different N/P ratios. p53 polyplex-loaded calcium pectinate (CP) microbeads were prepared by ionotropic gelation of pectin with calcium chloride and coated with Eudragit® S100. In vitro release studies showed that enteric-coated CP microbeads protected the release of p53 polyplex in upper GIT with less than 10% release. In-vitro cell line studies and in vivo studies in rat showed that polymethacrylate carrier could transfect the pDNA effectively. Results of in vivo gene expression study further confirmed the ability of enteric-coated calcium pectinate microbeads to deliver pDNA specifically to rat colon. Conclusively, enteric-coated calcium pectinate microbeads released p53 polyplex specifically in colon and could serve as an effective alternative for CRC therapy.     

响应面法优化肉苁蓉松果菊苷肠溶微球的制备

目的 制备肉苁蓉松果菊苷肠溶微球,筛选最佳制备工艺,并考察其体外释放特性。方法 用离子凝胶-干燥法制备肉苁蓉松果菊苷肠溶微球,以包封率为考察指标,用响应面法优化其制备工艺。结果 制备肉苁蓉松果菊苷肠溶微球最佳工艺参数：海藻酸钠浓度36.33 mg/ml、氯化钙浓度10.82 mg/ml、壳聚糖浓度10.93 mg/ml。结论 优选的工艺稳定、可行,制备的微球包封率高、缓释效果较好。     

Flicking technique for microencapsulation of cells in calcium alginate leading to the microtissue formation

Microbeads have wide applications in biomedical engineering field that include drug delivery, encapsulation of biomolecules, tissue padding and tissue regeneration. In this paper, we report a simple, yet efficient, flicking technique to produce microcapsules of calcium alginate at a narrow distribution of size. The system consists of an infusion pump and a customised flicker that taps the syringe needle for dispersing microcapsules of sodium alginate that polymerised in the calcium chloride solution. The flow rate of the syringe pump and the velocity of the flicker were studied to achieve a well controlled and tunable size distribution of microbeads ranging from 200 to 400?μm. At a flow rate of 4?μl/min and flicking rate of 80?rpm, a narrow size distribution of microbeads were produced. Via this technique, HaCaT cells were encapsulated in calcium alginate microbeads that grown into microtissues with a size ranging from 100 to 300?μm after two weeks of culture. These microtissues could be potentially useful for pharmacological application.     

Release behaviour of diclofenac sodium dispersed in Gelucire® and encapsulated with alginate beads

Sustained release polymeric particles containing diclofenac sodium dispersed in Gelucire® matrix and encapsulated in calcium alginate shell were prepared with different drug-to-polymer ratios and also with different concentrations of sodium alginate for a fixed drug-to-polymer ratio in an aqueous environment. Spherical particles were formed by dropping an emulsion of diclofenac sodium in Gelucire® matrix, emulsified with sodium alginate, into calcium chloride solution. The gelled beads formed by ionotropic gelation of alginate with calcium ions showed sustained release of the water soluble drug in in-vitro release study. Drug release was a function of square-root of time, suggesting a matrix diffusion release pattern. The rate of release was significantly suppressed with increasing proportions of Gelucire® in the mixture. Sustained and complete release was achieved with Gelucire® of low melting point and low HLB value. No significant drug release occurred in a dissolution medium of pH 1.5, whereas complete release was observed at pH 6.8, consistent with considerable swelling of the alginate gel at this pH.     

Treatment of Radix Dipsaci extract prevents long bone loss induced by modeled microgravity in hindlimb unloading rats

Abstract

Context: Radix Dipsaci is a kidney tonifying herbal medicine with a long history of safe use for treatment of bone fractures and joint diseases in China. Previous studies have shown that Radix Dipsaci extract (RDE) could prevent bone loss in ovariectomized rats.

Objective: This study investigates the effect of RDE against bone loss induced by simulated microgravity.

Materials and methods: A hindlimb unloading rat model was established to determine the effect of RDE on bone mineral density and bone microarchitecture. Twenty-four male Sprague–Dawley rats were divided into four groups (n?=?6 per group): control (CON), hindlimb unloading with vehicle (HLU), hindlimb unloading treated with alendronate (HLU-ALN, 2.0?mg/kg/d), and hindlimb unloading treated with RDE (HLU-RDE, 500?mg/kg/d). RDE or ALN was administrated orally for 4 weeks.

Results: Treatment with RDE had a positive effect on mechanical strength, BMD, BMC, bone turnover markers, and the changes in urinary calcium and phosphorus excretion. MicroCT analysis showed that RDE significantly prevented the reduction of the bone volume fraction, connectivity density, trabecular number, thickness, tissue mineral density, and tissue mineral content as well as improved the trabecular separation and structure model index.

Discussion and conclusion: RDE was demonstrated to prevent the loss of bone mass induced by HLU treatment, which suggests the potential application of RDE in the treatment of microgravity-induced bone loss.     

In vitro survival of Bifidobacterium bifidum microencapsulated in zein-coated alginate hydrogel microbeads

The Bifidobacterium bifidum susceptibility in gastrointestinal conditions and storage stability limit its use as potential probiotics. The current study was design to encapsulate B. bifidum using sodium alginate (SA, 1.4% w/v) and different concentration of zein as coating material, that is, Z₁ (1% w/v), Z₂ (3% w/v), Z₃ (5% w/v), Z₄ (7% w/v), Z₅ (9% w/v). The resultant microbeads were further investigated for encapsulation efficiency, survival in gastrointestinal conditions, release profile in intestinal fluid, storage stability and morphological characteristics. The highest encapsulation efficiency (94.56%) and viable count (>10⁷ log CFU/g) was observed in Z₄ (7% w/v). Viable cell count of B. bifidum was >10⁶ log CFU/g in all the zein-coated microbeads as compare to free cells (10³ log CFU/g) and SA (10⁵ log CFU/g) at 4 °C after 32 days of storage. Therefore, B. bifidum encapsulated in zein-coated alginate microbeads present improved survival during gastric transit and storage.     

PREVENTIVE NUTRITIONAL FACTORS IN EPIDEMIOLOGY: INTERACTION BETWEEN SODIUM AND CALCIUM

1. It is generally believed, though difficult to prove, that diet plays a role in the risk of various diseases. Components of difficulties include several issues such as dietary assessment method, regression dilution bias, multicolinearity and interaction among nutrients. 2. The present study focuses on colinearity and interaction between sodium and calcium, which should be cautiously examined in nutritional epidemiological studies in relation to blood pressure and bone mineral density. 3. The World Health Organization's International Cooperative Cardiovascular Diseases and Alimentary Comparison study showed significant multicolinearity among urinary sodium, calcium and urea nitrogen as well as urinary calcium and magnesium. Urinary sodium and calcium had significant correlation (r = 0.438, P < 0.05, n = 48) by cross-centre analysis. 4. Interaction between sodium and calcium on bone mineral density is studied using the data set from bone mineral density screening for 1658 females, aged 20-40 years, in Yokohama, Japan. Among those who have lower calcium intake (< 600 mg/day), higher calcium intake (%) from small fish, which is likely to be associated with a high salt diet, related to significantly lower mineral bone density. 5. Interaction between sodium and calcium on bone mineral density among young Japanese females is suggested. Moderate sodium restriction is needed for prevention of not only cardiovascular diseases but also osteoporosis.     

Effect of alginate composition and purity on alginate microspheres

Background: Alginate is commonly used to microencapsulate islets in experiments with islet allografts and xenografts for the treatment of Type I diabetes. The purpose of the present study is to determine the effects of alginate composition and purity on the morphology and size of microspheres. Methods: Microcapsules produced with the impure alginate types, medium-viscosity high-guluronic acid (IMVG), low-viscosity high-G (ILVG), low-viscosity high-mannuronic acid (ILVM) and medium-viscosity high-M (IMVM) were compared with one another and others generated with a highly purified LVM (HPLVM) alginate. Droplets of 1.5% alginate from an air-syringe pump were gelled in 1.1% CaCl₂ solution. While leaving the alginate pressure and needle recess constant, the air-jacket pressure was varied between 9.5–10.5 PPSI to enhance stable microcapsule generation and different batches of microbeads were made from each alginate type. Results: The sizes of the high-guluronic acid alginate microbeads were consistently bigger than those of the corresponding high-mannuronic acid alginate beads at all air-jacket settings. At the optimal air-jacket pressure of 9.0 PPSI, the mean+SD diameter of the IMVG microbeads was 780+20?µm, while that of IMVM was 607+44?µm (p<0.0001, n?=?30). Similarly, the mean ILVG microbead diameter was 816+28 µm compared to 656+26?µm for ILVM capsules (p<0.0001, n?=?30). Less polymorphism was found with the HPLVM microspheres than with the ILVM microbeads. Conclusion: Highly purified high-mannuronic acid alginate will provide smaller, spherical microcapsules suitable for islet cell transplantation.     

Chitosan–sodium alginate blended polyelectrolyte complexes as potential multiparticulate carrier system: colon-targeted delivery and gamma scintigraphic imaging

Objectives: The objective of this study is to develop stable, biodegradable chitosan–sodium alginate-based dual, ionic cross-linked multiparticulate system (microbeads) of tinidazole for targeted colon delivery and sustained drug release for the treatment of amoebiasis and thereby evaluating its targeting approach through in vivo gamma scintigraphic imaging technique.

Methods: The chitosan–sodium alginate-based multiparticulate system developed was producing sustained effect by virtue of its mechanical strength using double ionotropic gelation method utilizing calcium chloride and sodium sulfate as first and second cross-linkers respectively. Prepared formulations were evaluated for percent yield, drug entrapment efficiency, particle size, degree of swelling, in vitro kinetics, and in vivo targeting potentials using gamma scintigraphic imaging technique.

Results: The obtained particulates were spherical, free flowing, and had a mean particle size ranging from 1.422 mm to 1.881 mm, whereas percent yield and percent drug entrapment efficiency was found to be in between 72.61 to 82.43% and 63.25 to 79.32% respectively.

Conclusion: The prepared multiparticulate system showed better sustained release property and in vivo ability to target colon for drug delivery. Hence, the developed multiparticulate system could be a promising device to achieve greater site-specificity to colon.     

A dry powder formulation from silk fibroin microspheres as a topical auto-gelling device

With the aim of establishing the formulation of a new hydrophilic auto-gelling medical device for biomedical applications, fibroin-based microspheres were prepared. The proposed microspheres were produced by a cost-effective and industrially scalable technique, such as the spray-drying. Spray-dried silk fibroin microspheres were obtained and the effects of different hydrophilic polymer on the process yield, microsphere morphology and conformation transition of fibroin were evaluated. The final auto-gelling formulations were obtained by adding calcium gluconate (as a calcium source for alginate crosslinking) to the prepared microspheres and tested by an in vitro gelling test. This study showed that the combination of fibroin with sodium alginate and poloxamer produced the most promising auto-gelling formulation for specific biomedical applications, such as the treatment of pressure ulcers.     

Compaction Properties of Composite Particles Consisting of Lactose with Sodium Alginate Prepared by Spray-Drying

Purpose. Composite particles of lactose with a small amount of sodium alginate were prepared by spray-drying (SD) in an effort to improve the compactibility of the polymer for direct compression. The compaction behavior of the SD composite particles with a range of polymer contents was investigated. Methods. Composite particles were prepared by spray-drying an aqueous solution of lactose and sodium alginate at various formulating ratios. Improvement in the compactibility of the composite particles was evaluated by measuring the tablet tensile strength, porosity-applied pressure profiles, stress relaxation, elastic recovery of the compressed powder, and surface properties of the tablets by scanning electron microscopy. Results. The tensile strength of compacts formed from the SD composite particles containing sodium alginate (10 wt%) was as high as that of spray-dried amorphous lactose. The improved compaction was attributed to the higher relaxation pressure and lower elastic recovery of the composite particles compared with -lactose monohydrate. However, increasing the sodium alginate content of the SD composite particles above 10 wt% led to a marked reduction in the tensile strength of the resultant tablets. Scanning electron micrographs revealed that composite particles with a good compactibility fused totally in the tablets while composite particles containing 15% or more sodium alginate retained their shape, even after compression. The presence of sodium alginate layered uniformly on the surface of the particles and the increase in the glass transition temperature of the particles, possibly due to interpolation of sodium alginate are responsible for the reduction in the fusion property of the composite particles on compression. Conclusions. Although increasing the sodium alginate content of SD composite particles led to an increase in their plastic deformation, fusion on compression was prevented by the presence of sodium alginate. The reduced compactibility of SD composite particles with an excess amount of sodium alginate was attributed to reduced cohesion and fusion of the particles during compression.     

仙灵骨葆胶囊联合阿仑膦酸钠对骨质疏松症患者的骨代谢及骨转换指标的影响

目的 探讨仙灵骨葆胶囊联合阿仑膦酸钠对骨质疏松症患者骨代谢及骨转换指标的影响。方法 选取2015年9月—2018年9月西宁市第一人民医院92例骨质疏松症患者作为研究对象,按照随机数字法将患者分为对照组和观察组,每组各46例。对照组患者口服阿仑膦酸钠片,10 mg/次,1次/d。观察组在对照组的基础上口服仙灵骨葆胶囊,2粒/次,3次/d,两组均持续治疗3个月。观察两组患者的临床疗效,同时比较两组治疗前后的骨代谢、骨转换指标及腰椎L_2-4和股骨颈骨密度。结果 治疗后,观察组总有效率为93.48%,对照组总有效率为78.26%,两组总有效率比较差异具有统计学意义（P<0.05）。治疗后,两组患者骨源性碱性磷酸酶（BAP）、骨钙素（BGP）和碱性磷酸酶（ALP）水平均显著升高,24 h尿钙（U-Ca）水平显著降低（P<0.05）,且观察组骨代谢各指标水平显著优于对照组（P<0.05）。治疗后,两组患者的核因子κB受体活化因子配基（RANKL）及Ⅰ型胶原交联C-末端肽（CTX-1）水平均显著降低,骨保护素（OPG）水平显著升高（P<0.05）,且观察组各骨转换指标水平显著优于对照组（P<0.05）。两组治疗后腰椎L_2-4和股骨颈骨密度均显著增加（P<0.05）,且观察组腰椎L_2-4和股骨颈骨密度显著高于对照组（P<0.05）。结论 仙灵骨葆胶囊联合阿仑膦酸钠可有效改善患者的骨代谢、骨转换指标及骨密度水平,疗效确切,在骨质疏松症患者治疗中具有较高的应用价值。     

Formulation optimization and <Emphasis Type="Italic">in vivo</Emphasis> evaluation of mucoadhesive xyloglucan microspheres

Mucoadhesive microspheres of the novel polymer, xyloglucan, have been formulated and their performance characteristics have been systematically evaluated in vitro and in vivo. The mucoadhesive microspheres were obtained by incorporating glipizide as model drug in xyloglucan as a mucoadhesive polymer and sodium alginate as a gel-forming polymer by the orifice-ionic gelation method. A3² factorial design was employed to study the effect of independent variables, xyloglucan concentration (X ₁ ) and calcium chloride (CaCl₂) concentration (X ₂ ), on the dependent variables including drug entrapment efficiency, release time (t ₈₀), and percentage mucoadhesion in 1h. The best batch exhibited high drug entrapment efficiency (92.98%) and percentage mucoadhesion (78% after 1h). The drug release was also controlled for more than 8 hours. In vivo testing of the mucoadhesive microspheres revealed significant hypoglycemic effect of glipizide.     

Statins: under investigation for increasing bone mineral density and augmenting fracture healing

Background: Statins are 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors and have been shown to possess anti-lipidaemic properties effective in lowering cholesterol. Recent evidence has suggested beneficial pleiotropic effects, including that of fracture healing, alongside its widely accepted ability to reduce the incidence of cardiovascular disease. Objectives: A comprehensive review of the recent literature on the effect of statins on bone mineral density and fracture healing. Methods: Medline/Ovid and EMBASE search and manual search of bibliography of key papers, on the effects of statins on bone metabolism including in vitro and in vivo studies, as well as clinical trials on the effects of statins on bone mineral density and fracture risk. Results/conclusions: There is robust in vitro and in vivo evidence to suggest the anabolic effects of statins on bone metabolism. Although evidence in patients with osteoporosis is conflicting, several studies have shown that the use of statins is associated with increases in bone mass density and reduction in fracture risk. Conflicting studies identified may be due to different routes of administration, types of statins employed and low doses used. Taken together, there is strong evidence to suggest that statins have beneficial effects on fracture healing that would support further clinical trials investigating such properties.     

Alginate/Poly-L-Lysine Microparticles for the Intestinal Delivery of Antisense Oligonucleotides

Purpose. A microparticle carrier based on alginate and poly-L-lysine was developed and evaluated for the delivery of antisense oligonucleotides at the intestinal site. Formulations of oligonucleotide-loaded microparticles having differences in the carrier molecular weight and composition were characterized in vitro and in vivo. Methods. Polymeric microparticles were prepared by ionotropic gelation and crosslinking of alginate with calcium ions and poly-L-lysine. The loading of the antisense oligonucleotide into the microparticles was achieved by absorption in aqueous medium. The association capacity, loading and particle size of the microparticles were characterized. The in vivo performances of various formulations after intrajejunal administration were studied in rat and in dog models. Results. Microparticles had a sponge-like structure and an oligonucleotide loading of 27-35%. The composition of the medium affected the particle size and the in vitro release profiles. The oligonucleotide bioavailability after intrajejunal administration to rats in the presence of permeation enhancers was good for most of the tested systems. The application of microparticles in powder form compared to an equivalent suspension improved the intrajejunal bioavailability of the oligonucleotide (25% and 10% respectively) in rats. On the contrary, the intrajejunal administration to dogs resulted in poor oligonucleotide bioavailability (0.42%). Conclusions. The formulation of antisense oligonucleotides within alginate and poly-L-lysine microparticles is a promising strategy for the oral application.     

Encapsulation of olive leaf antioxidants in microbeads: Application of alginate and chitosan as wall materials

Edible microcapsule technology has been declared as a newly developed technology in 21st century by some certain authorities in order to preserve food products. Encapsulation of the bioactive materials in edible coatings is a blessing that can eliminate many undesirable situations that might arise when it is used as additive. In this study, olive leaf extract has been evaluated as active material to prepare microcapsules by using alginate as coating. Ionic gelation was used to produce microbeads. The experimental design of the encapsulation system, the effects of the process parameters, the modeling of the experimental data and the optimization of the conditions were carried out with Box-Behnken design of response surface method (Box-Behnken-RSM). Box-Behnken-RSM produced 17 experimental runs. Calcium chloride (2–15%, w/v) and sodium alginate concentrations (1–2%, w/v), and hardening time (15–45 min) were selected as independent variables, while encapsulation efficiency (EE) of the capsules in terms of total phenolic content (TPC) and oleuropein concentration were responses. Impact of chitosan as coating layer was also investigated with three different ratios of chitosan (0.4%, 0.7%, 1% w/v). Accelerated oxidation test was employed to measure the stability of the microcapsules against oxidation by means of Rancimat method. Encapsulation of the olive leaf extract in alginate microbeads was satisfying with >70% and >90% efficiencies with respect to TPC and oleuropein under optimum conditions (2.34% calcium chloride concentration and 2% sodium alginate for 26 min of hardening time).     

聚合多巴胺改性紫杉醇纳米粒靶向治疗乳腺癌骨转移

目的 基于多巴胺衍生物的粘附性,构建表面改性的紫杉醇（PTX）纳米粒,连接阿伦磷酸钠（ALN）,对构建的新型纳米粒进行表征并考察其对鼠源性乳腺癌4T1细胞的毒性作用。方法 采用溶剂沉淀法制备PTX纳米粒,将纳米粒放置于0.5 mg/mL的盐酸多巴胺Tris缓冲盐溶液中,在表面形成聚合多巴胺（PDA）,随后与亲骨性药物ALN结合,得到新型PTX-PEG₅₀₀₀PCL₁₀₀₀-PDA-ALN纳米粒。ZS型电位仪测定纳米粒的粒径、粒度分布和表面电位;透射电镜观察形态;考察其在不同介质中的稳定性、溶血性;考察ALN用量及钙离子存在条件对新型纳米粒吸附羟基磷灰石[（Ca₁₀PO₄）₆（OH）₂,HA]的影响;研究其体外抗4T1乳腺癌细胞活性。结果 成功制得PTX-PEG₅₀₀₀PCL₁₀₀₀-PDA-ALN纳米粒,棒状,粒径为（178.5±2.3）nm,分散指数（PDI）值为0.213±0.06,Zeta电位为（20.12±2.45）mV;在5%葡萄糖、0.9%生理盐水、血浆中基本稳定;无溶血现象;随着ALN用量增加,新型纳米粒对HA吸附率增大,可达48.63%,游离钙离子降低其对HA的吸附能力;MTT结果显示,其对4T1乳腺癌细胞发挥显著抑制作用。结论 制备的PTX-PEG₅₀₀₀PCL₁₀₀₀-PDA-ALN纳米粒粒度分布均匀、稳定性良好、不溶血;通过ALN与HA上的钙离子相结合,亲骨性良好;对4T1细胞发挥毒性作用。     

Formation of alginate microspheres produced using emulsification technique

This study investigated the formative process of alginate microspheres produced using an emulsification technique. The alginate microspheres were produced by cross-linking alginate globules dispersed in a continuous organic phase using various calcium salts: calcium chloride, calcium acetate, calcium lactate and calcium gluconate. The size, shape, drug content and Ca²⁺ content of the microspheres were evaluated. The tack, viscosity and pH of the calcium salt solution and percentage of Ca²⁺ partitioned into the organic phase were determined. Microscopic examination of the test emulsion at various stages of the emulsification process was also carried out. The propensity of cross-linking reaction was found to be dependent on successful collision between alginate and calcium salt globules. Examination of the characteristics of microspheres indicated that the formed microsphere was a resultant product of alginate globule clustering. The growth propensity of microspheres was promoted by the higher rate and extent of cross-linkage which was governed by the pH, tack and/or Ca²⁺ content of the cross-linking solution, as well as the dissociation constant and diffusivity of the calcium salt. Overall, the amount of free Ca²⁺ cross-linked with alginate in the formed microspheres was in the following order: calcium acetate > calcium chloride + calcium acetate > calcium chloride + calcium gluconate; calcium chloride + calcium lactate > calcium chloride. In microencapsulation by emulsification, the mean size of the microspheres produced can be modified by varying the tack, pH and Ca²⁺ content of the cross-linking solution and through the use of a combination of calcium salts. The shape of the microspheres produced was, nonetheless, unaffected by the physicochemical properties of the cross-linking solution.     

The effect of antibiotics on bone healing: current evidence

Introduction: Bone healing is a complex cascade of events that involves the proliferation and differentiation of osteoblastic cells under the influence of signals from growth factors, cytokines and mechanical loading. Several medications have been found to interact negatively with this process including cytostatics, NSAIDs and corticosteroids; however, the effect of antibiotics on bone repair processes remains obscure.

Areas covered: The authors offer a comprehensive review of the existing literature on the in vivo and in vitro effect of antibiotics on bone, bone cells and fracture healing. The authors describe the pharmacokinetic characteristics of antibiotics after parenteral administration as well as their levels when applied locally together with a delivery vehicle.

Expert opinion: The available experimental data and clinical evidence are rather limited to allow safe conclusions. In vitro studies indicate that high doses administered after systemic administration have little or no direct effect on bone cells. Further studies are desirable to define the effect of higher or prolonged concentrations on bone biology and especially that of high concentrations released by locally implanted antibiotic-delivery systems, that is, bone cement, spacers and beads.